Phospholipid scramblase 3

ABSTRACT

Phospholipid scramblase 3 (PLS3) is a newly recognized member of a family of proteins responsible for phospholipid translocation between two lipid compartments. A novel isoform of PLS3 is identified and characterized herein. The function of PLS3 in mitochondria was disrupted, yielding an inactive mutant PLS3(F258V). Cells transfected with PLS3(F258V) exhibited reduced proliferative capacity that was unaffected by the presence of Na 3 N. PLS3(F258V)-expressing cells exhibit abnormal mitochondrial metabolism and structure and were associated with decreased sensitivity to UV- and tBid-induced apoptosis, and diminished translocation of cardiolipin to the outer mitochondrial membrane. Cells transfected with wild-type PLS3 displayed increased sensitivity to apoptosis and enhanced cardiolipin translocation. These studies identify PLS3 as a regulator of mitochondrial structure and respiration, and cardiolipin transport in apoptosis.

BACKGROUND OF THE INVENTION

Regulation of apoptosis, or programmed cell death, is critical fordevelopment and tissue homeostasis. Dysregulation of apoptosis is a keyfactor in neoplastic transformation. Reed, Dysregulation of apoptosis incancer, J. Clin. Oncol., 17: 2941-2953, (1999); Ionov et al., Mutationalinactivation of the proapoptopic gene BAX confers selective advantageduring tumor clonal evolution, Proc. Natl. Acad. Sci. USA., 97:10872-10877, (2000). Apoptotic cell death is characterized by aproteolytic caspase cascade that emanates from either an ‘extrinsic’pathway, initiated by membrane-bound death receptors leading toactivation of caspase-8, or an ‘intrinsic’ pathway triggered byDNA-damaging drugs and UV radiation leading to mitochondrialdepolarization and subsequent activation of caspase-9. Green and Evan, Amatter of life and death, Cancer Cell, 1: 19-30, (2002). Caspaseactivation leads to distinct morphological changes, includingmitochondrial disintegration, followed by nuclear fragmentation,chromatin condensation, and cytoplasmic membrane blebbing. Cory andAdams, Matters of life and death: programmed cell death at Cold SpringHarbor, Biochim. Biophys. Acta, 1377: R25-R44, (1998); Kroemer and Reed,Mitochondrial control of cell death, Nat. Med., 6: 513-519, (2000).

An additional early morphological event in cells undergoing apoptosis istranslocation of phosphatidylserine (PS) from the inner to the outerleaflet of the plasma membrane. Fadok et al., Exposure ofphosphatidylserine oil the surface of apoptotic lymphocytes triggersspecific recognition and removal by macrophages, J. Immunol, 148:2207-2216, (1992); Bratton et al., Appearance of phosphatidylserine onapoptotic cells requires calcium-mediated nonspecific flip-flop and isenhanced by loss of the aminophospholipid translocase, J. Biol. Chem.,272: 26159-26165 (1997); and Martin et al., Early redistribution ofplasma membrane phosphatidylserine is a general feature of apoptosisregardless of the initiating stimulus: inhibition by overexpression ofBcl-2 and Abl, J. Exp. Med., 182: 1545-1556, (1995). Externalized PS isrecognized by macrophages, which rapidly remove apoptotic cells byphagocytosis. Fadok et al., Loss of phospholipids asymmetry and surfaceexposure of phosphatidylserine is required for phagocytosis of apoptoticcells by macrophages and fibroblasts, J. Biol. Chem., 276: 10781-1077(2001); Fadok et al., A receptor for phosphatidylserine-specificclearance of apoptotic cells, Nature,405: 85-90 (2000). Mitochondria arecentral integrators of most apoptotic pathways. Brenner and Kroemer,Mitochondria—the death signal integrators, Science, 289: 1150-1151,(2000). Activation of the intrinsic pathway causes mitochondrial releaseof a number of pro-apoptotic molecules including cytochrome c,endonuclease G (Parrish et al., Mitochondrial endonuclease G isimportant for apoptosis in C. elegans, Nature, 412: 90-94 (2001); Li etal., Endonuclease G is an apoptotic DNase when released frommitochondria, Nature, 412: 95-99, (2001), SMAC/Diablo (Du et al., Smac,a mitochondrial protein that promotes cytochrome c-dependent caspaseactivation by eliminating IAP inhibition [In Process Citation], Cell,102: 33-42, (2000); Verhagen et al., Identification of DIABLO, amammalian protein that promotes apoptosis by binding to and antagonizingIAP proteins [In Process Citation], Cell, 102: 43-53 (2000)), andapoptosis-inducing factor (Susin et al., Molecular characterization ofmitochondrial apoptosis-inducing factor, Nature, 397: 441-446, (1999)).

In addition, multiple pro-apoptotic regulators including Bax, Bad, Bid,Bim, p53, JNK, PKC-y, nuclear receptor TR3 (Brenner and Kroemer,Apoptosis. Mitochondria—the death signal integrators, Science, 289:1150-1151 (2000); Li et al., Cytochrome c release and apoptosis inducedby mitochondrial targeting of nuclear orphan receptor TR3, Science, 289:1159-1164 (2000)), and the Peutz-Jegher gene product LKB1 (Karuman etal., The Peutz-Jegher gene product LKB1 is a mediator of p53-dependentcell death, Mol. Cell, 7: 1307-1319, (2001)) are translocated tomitochondria during apoptosis. In some cell types, the extrinsic pathwayis linked to the intrinsic pathway via activation of caspase-8, whichcauses NH₂-terminal cleavage of Bid to generate tBid (Luo et al., Bid, aBcl2 interacting protein, mediates cytochrome c release frommitochondria in response to activation of cell surface death receptors,Cell, 94: 481-490, (1998)). The active tBid fragment is N-myristoylated(Zha et al., Posttranslational N-myristoylation of BID as a molecularswitch for targeting mitochondria and apoptosis, Science, 290:1761-1765, (2000)) and then localizes to mitochondria through a positiveinteraction with cardiolipin (CL; Lutter et al., Cardiolipinprovidesspecificity for targeting of tBid to mitochondria, Nat. Cell Biol., 2:754-761, (2000)). Activated tBid induces CL-dependent activation of Baxand Bak to form cytochrome c channels during apoptosis. Kuwana et al.,Bid, Bax, and lipids cooperate to form supramolecular openings in theouter mitochondrial membrane, Cell, 111: 331-342, (2002). In the absenceof Bax and Bak, tBid is unable to induce cytochrome c release. Wei etal., tBID, a membrane-targeted death ligand, oligomerizes BAK to releasecytochrome c, Genes Dev., 14: 2060-2071, (2000); Wei et al., ProaptopticBAX and BAK: a requisite gateway to mitochondrial dysfunction and death,Science, 292: 727-730, (2001); and Korsmeyer, Pro-apoptotic cascadeactivates BID, which oligomerizes BAK or BAX into pores that result inthe release of cytochrome c, Cell Death Differ., 7: 1166-1173, (2000).

Phospholipid scramblases (PLS) are enzymes responsible for bidirectionalmovement of phospholipids (Bevers et al., Lipid translocation across theplasma membrane of mammalian cells, Biochim. Biophys. Acta, 1439:317-330, (1999)), and four PLS family members have been identified(Wiedmer et al., Identification of three new members of thephospholipids scramblase gene family, Biochim. Biophys. Acta, 1467:244-253, (2000)). PLS1 is located in the plasma membrane and isresponsible for translocation of phospholipids between the inner andouter leaflets. Zhou et al., Molecular cloning of human plasma membranephospholipids scramblase, A protein mediating transbilayer movement ofplasma membrane phospholipids, J. Biol. Chem., 272: 18240-18244 (1997).Although apoptotic PS translocation was unaltered in PLS1-deficient mice(Zhou et al., Normal hemostasis but defective hematopoietic response togrowth factors in mice deficient in phospholipid scramblase 1, Blood,99: 4030-4038, (2002)), the role of PLS1 in apoptosis remains uncleargiven the presence of additional enzymes associated with plasma membranephospholipid translocation such as aminophospholipid translocase.(Bevers et al., Lipid translocation across the plasma membrane ofmammalian cells, Biochim Biophys. Acta, 1439: 317-330, (1999); Zhao etal., Level of expression of phospholipids scramblase regulates inducedmovement of phosphatidylserine to the cell surface, J. Biol. Chem., 273:5503-5505, (1998); and Williamson et al., Phospholipid scramblaseactivation pathways in lymphocytes, Biochemistry, 40: 8065-8072 (2001).

PLS family members contain a conserved calcium-binding motif, and Zhouet al. (Identity of a conserved motif in phospholipids scramblase thatis required for Ca2+-accelerated transbilayer movement of membranephospholipids, Biochemistry, 37: 2356-2360, (1998)) found that mutationof residues in this region of PLS1 completely eliminated enzymaticactivity. PLS1 is phosphorylated at Thr-161 by PKC-8, which translocatesto the plasma membrane during apoptosis; in addition, PLS1 isphosphorylated at Tyr-69/74 by c-Abl kinase. Sun et al., c-Abl tyrosinekinase binds and phosphorylates phospholipids scramblase 1, J. Biol.Chem., 276: 29894-28990, (2001). The role of calcium binding and thesevarious phosphorylation events in PLS1 activation and regulation,however, remain to be determined.

A newly identified member of the scramblase family, designated PLS3, islocalized to the mitochondria rather than plasma membrane. Liu et al.,Phospholipid scramblase 3 is the mitochondrial target of protein kinaseC δ-induced apoptosis, Cancer Res., 63: 1153-1156, (2003). However,little is known regarding the physiological function of PLS3 inmitochondria. It has been shown previously that PLS3, like PLS1, isphosphorylated by PKC-δ. Id. A mitochondrial targeted PKC-δ dramaticallyenhanced susceptibility to apoptosis in cells overexpressing PLS3,suggesting that PLS3 is the direct mitochondrial effector ofPKC-δ-induced apoptosis. Id.

Members of the PKC family of kinases play diverse roles in cellsignaling, proliferation, apoptosis, and other cellular processes.Parker and Dekker, Protein Kinase C., Georgetown: Landes Bioscience,1997; and Ohno, The distinct biological potential of PKC isotypes,Protein Kinase C., pp. 75-95, Georgetown: Landes Bioscience, 1997. PKC-δis particularly associated with apoptosis. Denning et al., Caspaseactivation and disruption of mitochondrial membrane potential during UVradiation-induced apoptosis of human keratinocytes requires activationof protein kinase C., Cell. Death Differ., 9: 40-52, 2002; Fujii et al.,Involvement of protein kinase Cδ (PKCδ) in phorbol ester-inducedapoptosis in LNCαP prostate cancer cells, Lack of proteolytic cleavageof PCKδ, J. Biol. Chem., 275: 7574-7582 (2000); Li et al., Proteinkinase Cδ targets mitochondria, alters mitochondrial membrane potential,and induces apoptosis in normal and neoplastic keratinocytes whenoverexpressed by an adenoviral vector, Mol. Cell. Biol., 18: 8574-8558(1999); Majumder et al., Mitochondrial translocation of protein kinaseCδ in phorbol ester-induced cytochrome c release and apoptosis, J. Biol.Chem., 275: 21793-21796 (2000); Mandil et al., Protein kinase Cα andprotein kinase Cδ play opposite roles in the proliferation and apoptosisof glioma cells, Cancer Res., 61: 4612-4619 (2001); and Yoshida andKufe, Negative regulation of the SHPTP1 protein tyrosine phosphatase byprotein kinase Cδ in response to DNA damage, Mol. Pharmacol., 60:1431-1438 (2001). PKC-δ is present in the cytoplasm, and translocates tovarious organelles, including the nucleus, mitochondria, and the plasmamembrane, on apoptotic stimulation. Id. One substrate of PKCδ is PLS1 inthe plasma membrane. Frasch et al., Regulation of phospholipidsscramblase activity during apoptosis and cell activation by proteinkinase Cδ, J. Biol. Chem., 275: 23065-23073 (2000). However, theconsequence of PLS1 phosphorylation and how PLS1 contributes toapoptosis remain elusive. PKC-δ is activated during apoptosis bycaspase-mediated cleavage to become catalytically active. Ghayur, etal., Proteolytic activation of protein kinase C δ by an ICE/CED 3-likeprotease induces characteristics of apoptosis, J. Exp. Med.,184:2399-2404 (1996). This catalytic active fragment of PKC-δtranslocates to the mitochondria to induce apoptotic events. Inhibitionof PKC-δ blocks the disruption of the mitochondrial transmembranepotential. These results indicate that although PKC-δ plays an importantrole in triggering apoptosis through a mitochondrion-dependent pathway.However, the substrate of PKC-δ—in the mitochondria remainsunidentified.

The function of PLS is to associate translocated phospholipidsbidirectionally between two compartments. Bevers et al., Transmembranephospholipids distribution in blood cells: control mechanisms andpathophysiological significance, Biol. Chem., 379: 973-986 (1998) andSims and Wiedmer, Unraveling the mysteries of phospholipids scrambling,Thromb. Haemost., 85: 266-275 (2001). PLS1 translocates phospholipidsbetween the inner and outer leaflets of the plasma membrane, which isasymmetric in lipid composition. Although PS is translocated to theouter leaflet during apoptosis, it is still unclear whether PLS1 isresponsible for this translocation. Fadok et al., Exposure ofphosphatidylserine on the surface of apoptotic lymphocytes triggersspecific recognition and removal by macrophages, J. Immunol., 148:2207-2215 (1992); Fadeel et al., Phosphatidylserine exposure duringapoptosis is a cell-type-specific event and does not correlate withplasma membrane phospholipids scramblase expression, Biochem. Biophys.Res. Commun., 266: 504-511 (1999); Bratton et al., Appearance ofphosphatidylserine on apoptotic cells requires calcium-mediatednonspecific flip-flop and is enhanced by loss of the aminophospholipidtranslocase, J. Biol. Chem., 272: 26159-26165 (1997); Martin et al.,Early redistribution of plasma membrane phosphatidylserine is a generalfeature of apoptosis regardless of the initiating stimulus: inhibitionby overexpression of Bcl-2 and Abl., J. Exp. Med., 182: 1545-1556(1995); and Zhou et al., Molecular cloning of human plasma membranephospholipids scramblase. A protein mediating transbilayer movement ofplasma membrane phospholipids, J. Biol. Chem., 272: 18240-18244 (1997).

Bcl-B is the human orthologue of mouse BOO/Diva, a member of Bcl-2family similar to avian NR13. Avian NR13 was originally identified as av-src-activated gene (Gillet et al. 1995), and overexpression of NR13protein protected Baf-3 cells from IL-3 withdrawal-induced apoptosis(Mangeney et al. 1996). NR13 plays a major role in regulation of chickenbursal apoptosis. NR13 protected a bursa-derived cell line, DT40, fromserum-deprivation-induced apoptosis, and the level of NR13 correlatedwith bursal cell survival in vivo. Using a bursal transplantation model,it was demonstrated that one physiological function of NR13 is toprotect the bursal stem cells from programmed elimination (Lee et al.1999). Searching for mammalian homologues of avian NR13, two groupsidentified mouse BOO/Diva through EST database analysis (Inohara et al.1998; Song et al. 1999). It has a unique tissue distribution, expressedin all embryonic tissues but only in adult ovary. Protection from orpromotion of cell death by BOO/Diva was cell-type dependent. BOO/Divainteracted with Apaf-1; however, the physiological significance of thisinteraction is unclear (Inohara et al. 1998; Song et al. 1999). Bcl-B ishighly conserved between mouse and human, and its anti- or pro-apoptoticeffect is significantly weaker compared with Bcl-2 or Bcl-xL. Bcl-Binteracts with Bcl-2, Bcl-xL and Bax, but not Bak (Ke et al. 2001).Bcl-B is not completely localized in mitochondria, but translocates tomitochondria upon induction of cell death by microtubule-interferingagents. Bcl-B gene was mapped in chromosome 15q21, and deletion wasidentified in some primary cervical cancers (Lee et al. 2001).

The biochemical functions of Bcl-2 family in mitochondria are far fromclear. The crystal structure of Bcl-xL (Muchmore et al. 1996) suggestedthat Bcl-2 family members could form channels, and regulate themitochondrial transmembrane potential (Δψ) and the release of cytochromec upon pro-apoptotic stimulation (Shimizu et al. 1999; Vander Heiden andThompson 1999; Shimizu et al. 2000; Shimizu and Tsujimoto 2000). Theregulation of mitochondrial transmembrane potential (Δψ) is mediatedthrough a transmembrane permeability pore complex (Shimizu et al. 1999).This multiprotein complex contains many components, includingvoltage-dependent anion channel (VDAC) and peripheral benzodiazepinereceptor (PBR) on the outer membrane of mitochondria, adenine nucleotidetranslocator (ANT) on the inner membrane, and a matrix proteincyclophilin D (CphD). Shimizu et al. used liposomes carrying VDAC toshow that Bax and Bak accelerated the opening of VDAC, while Bcl-xLclosed VDAC through direct interaction with VDAC.

Using a BH4 domain peptide of Bcl-xL, they effectively preventedapoptotic cell death induced by etoposide treatment, indicating that BH4domain of Bcl-xL was critical for this function (Shimizu et al. 2000).On the other hand, the BH3-only Bcl-2 family members induce release ofcytochrome c without Δψ loss, and do not directly modulate VDAC activity(Shimizu and Tsujimoto 2000). These results demonstrated two differentfunctions of the mitochondrial transmembrane pore complex, i.e.maintaining mitochondrial transmembrane potential (Δψ) and serving as atransmembrane protein channel, and they are regulated through differentdomains of Bcl-2 family members.

Cardiolipin has recently drawn attention as a mitochondrial phospholipidinvolved in apoptosis. This dimeric phospholipid contains twophosphatidyl molecules linked by a glycerol moiety, thus in total fourfatty acyl chains and two phosphate groups (Hoch 1992). Cardiolipin ismainly located at the inner membranes of mitochondria (Krebs et al.1979) and is required for oxidative phosphorylation and high energyelectron transport (Jiang et al. 2000; Schlame et al. 2000). Cardiolipininteracts with cytochrome c, and peroxidation of cardiolipin dissociatescytochrome c from cardiolipin (Shidoji et al. 1999; Nomura et al. 2000).

Studies of cardiolipin were carried out with a cardiolipin-specificfluorescence dye NAO, nonyl-acridine orange (Garcia Fernandez et al.2000). NAO has a fluorescence emission at 625 nm with 2:1 stoichiometricinteraction to cardiolipin, and at 525 nm with 1:1 interaction.Measuring the amount of cardiolipin can be done with NAO by flowcytometry analysis (Garcia Fernandez et al. 2000). When cells becomeapoptotic, NAO binding decreases secondary to peroxidation ofcardiolipin, a process that contributes to the release of cytochrome c(Shidoji et al. 1999). Another function of cardiolipin was recentlyidentified as the mitochondrial target for tBid, which is cleaved fromBid upon apoptotic stimulation and induces activation of caspase 9 alongwith cytochrome c and Apaf-1 (Lutter et al. 2000).

Apoptosis is a center of much attention in current efforts to establishmore effective tailored treatments of cancer, as well as many otherconditions, including neurodegenerative disorders, and disordersaffecting the immune system. Research efforts often center on locatingcellular targets for the regulation of apoptosis. Some benefit may beobtained from the discovery of targets and methods useful for inducingapoptosis or preventing it. In cancer, it is desirable to discover orprovide compounds and/or methods for regulating apoptosis in targetedcancer cells as a treatment method. Such compounds and methods haveproven elusive, however. Similarly, in neurodegenerative disorders anddisorders affecting the immune system, it would be beneficial to providecompounds and/or methods for arresting apoptotic processes that proceedin an uncontrolled fashion, causing damage to nervous tissue.

Such gene targets, and methods for inducing and preventing apoptosis areprovided herein.

BRIEF SUMMARY OF THE INVENTION

The present invention relates in part to a gene which is involved inprocesses associated with apoptosis. More specifically, the presentinvention relates to the phospholipid scramblase 3 gene (hereinafter“PLS3”) involved in the modification of mitochondrial membranes duringapoptosis.

Phospholipid scramblase 3 (PLS3) is a newly recognized member of afamily of proteins responsible for phospholipid translocation betweentwo lipid compartments. An alternatively-spliced form of PLS3 (hereinreferred to as “PLS3α”, SEQ ID NO: 2) was identified using yeasttwo-hybrid screening to search for binding partners of Boo/Diva, a Bcl-2family member. This PLS3 isoform encodes a translated product (SEQ IDNO: 1) that differs at the last 17 amino acids from the PLS3 isoformknown and found in GenBank (herein referred to as “PLS3β”, SEQ ID NO:4). The sequences of the translated products, SEQ ID NO: 1, SEQ ID NO: 3are compared with those of a mouse, drosophila, and C. elegans inFIG. 1. The longer message (PLS3β) is expressed in heart and skeletalmuscle; while the short transcript (PLS3α) is expressed in spleen,liver, kidney and placenta. See, e.g., FIG. 2.

In contrast to PLS1 that is present in the plasma membrane, both PLS3αand PLS313 are localized in mitochondria. Computer prediction suggeststhat they have two transmembrane domains rather than the singletransmembrane domain found in PLS1. Proteinase K digestion and epitopemapping were used to study the topology of native PLS3α of mouse livermitochondria. Without being limited to any one theory, these techniquesappeared to yield the conclusion that PLS3α crosses both mitochondrialinner and outer membranes. This topology suggests that PLS3 may beresponsible for transferring phospholipids between the two mitochondrialmembranes.

The conserved calcium-binding motif of PLS3 was disrupted in the studiesdiscussed herein to evaluate the function of PLS3 in mitochondria. Thisprocess yielded an inactive mutant PLS3(F258V). Cells transfected withPLS3(F258V) exhibited reduced proliferative capacity that was unaffectedby the presence of Na₃N. Mitochondrial analysis revealed thatPLS3(F258V)-expressing cells have decreased mitochondrial mass shown bylower cytochrome c and cardiolipin content, poor mitochondrialrespiration, and reduced oxygen consumption and intracellular ATP. Incontrast, wild-type PLS3-transfected cells exhibit increasedmitochondrial mass and enhanced respiration. Electron microscopicexamination revealed that the mitochondria in PLS3(F258V)-expressingcells have densely packed cristae, and are fewer in number and largerthan those in cells transfected with wild-type PLS3.

It was also found that the abnormal mitochondrial metabolism andstructure in PLS3(F258V)-expressing cells was associated with decreasedsensitivity to UV- and tBid-induced apoptosis, and diminishedtranslocation of cardiolipin to the outer mitochondrial membrane. Bycontrast, wild-type PLS3-transfected cells displayed increasedsensitivity to apoptosis and enhanced cardiolipin translocation. Thesestudies appear to identify PLS3 as a regulator of mitochondrialstructure and respiration, and cardiolipin transport in apoptosis.

In addition to the above, it was noted that the deletion mutant of PLS3disrupted mitochondrial transmembrane potential and induced cell death.Incubation of purified PLS3 protein with mitochondria in vitro resultedin disruption of mitochondrial transmembrane potential and cytochrome crelease. In phospholipid transfer assays, PLS3 was noted to have asubstrate specificity to cardiolipin. UV irradiation and overexpressionof PLS3 in 293 cells increase the percentage of cardiolipin at the outermembrane of mitochondria. Co-expression of Bcl-B and PLS3 enhanced thepro-apoptotic effect of Bcl-B. This pro-apoptotic effect of Bcl-B,however, is inhibited by an inactive mutant of PLS3. These resultsindicate that PLS3 is a downstream effector of Bcl-B.

These results also make PLS3 a target for research and discovery ofcompounds for the induction of apoptosis or for blocking apoptosis.Indeed, the discovery and isolation of such a gene allows the screeningof compounds to isolate molecules capable of up- or down-regulating theproduction of PLS3 in a cell, and thus inducing or blocking apoptosis.

PKC-δ translocates to mitochondria during apoptosis, but itsmitochondrial target remains unclear. It was found that PKC-δ physicallyinteracted with and phosphorylated phospholipid scramblase 3 (PLS3)after UV irradiation. PLS3 is a high affinity substrate for PKC-δ invitro with the km at 10.5 nM. Cells expressing wild-type PLS3 becameapoptotic upon phorbol ester stimulation; while the control cells didnot. Expression of a mitochondrial targeted PKC-δ enhanced apoptosismore prominently in HeLa-PLS3 cells than control HeLa cells and HeLacells expressing an inactive PLS3 mutant. These results indicate thatPLS3 is a downstream effector of PKC-δ in the mitochondria.

Thus, the invention provides a novel target for the regulation ofcellular apoptosis. In part, the invention provides a novel isoform ofPLS3, namely PLS3α (SEQ ID NO: 2, product SEQ ID NO: 1). The inventionfurther provides methods of rendering a cell resistant to apoptosis. Inaddition, the invention provides methods of sensitizing a cell toapoptosis. Each of these is discussed in greater detail below.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

In order that the manner in which the above-recited and other featuresand advantages of the invention are obtained will be readily understood,a more particular description of the invention briefly described abovewill be rendered by reference to specific embodiments thereof which areillustrated in the appended drawings. Understanding that these drawingsdepict only typical embodiments of the invention and are not thereforeto be considered to be limiting of its scope, the invention will bedescribed and explained with additional specificity and detail throughthe use of the accompanying drawings in which:

FIG. 1: Clustal alignment of PLS3 proteins of human (PLS3α, PLS3β),mouse, Drosophila and C. elegans. The amino acids that are identicalamong four species are marked by “*”. The amino acids that are similarare marked by “:”. The differences between PLS3α and PLS3β arehighlighted in bold.

FIG. 2: (a) Physical interaction of PLS3α with Bcl-B. 293T cell lysatesfrom PLS3α and EGFP-Bcl-B overexpressing cells were immunoprecipitatedwith polyclonal PLS3 antibody and subjected to Western analysis withEGFP monoclonal antibody. IP control was performed with pre-immuneserum. (b) Tissue distribution of PLS3. Northern analysis of PLS3 wasperformed in human 12-lane MTN blots with PLS3 cDNA as probe. The probewith 2×10⁶ cpm/ml activity was used for hybridization. PLS3 blots wereexposed for 2 days, and actin control was exposed for 12 hours. Twoarrows indicate the two alternatively spliced forms of PLS3. The loweris PLS3α and the upper is PLS3β.

FIG. 3. Subcellular localization of PLS3 with EGFP-PLS3 fusion protein.(a) EGFP-PLS3α was transfected into 293 cells, and MitoTracker dye wasthen added for localization. Cells were visualized under confocalmicroscope, revealing that EGFP-PLS3α corresponds with MitoTracker.EGFP-PLS3β exhibits a similar pattern (not shown). (b) EGFP-ΔPLS3 wastransfected into 293 cells for colocalization with MitoTracker similarto (a). Three cells that express EGFP failed to pick up the redMitoTracker dye. (c) Subcellular fractionation for Western analysis withPLS3 polyclonal antibody. Cells with PLS3α overexpression were harvestedand subjected to differential centrifugation to obtain nuclei,mitochondria, microsomes and cytosols. Equal amounts of protein (20 μg)were loaded into gel for separation, and Western analysis was done withPLS3 antibody. Mitochondrial proteins from mouse liver and 293 cellswere also analyzed with the same antibody, with purified PLS3 used as apositive control.

FIG. 4. Topology of PLS3 in mitochondria. (a) Six potential topologiesof PLS3 protein in mitochondria are illustrated. The arrow direction isfrom N to C terminus of the protein. The solid bars represent antibodyrecognition sites. TM: transmembrane domain. (b) Western analysis ofmitochondria after protease K digestion with PLS3 Ab-1 (left) or Ab-N(right). The sizes of PLS3 or its cleavage products are indicated by MWmarkers. (c) Mitoplasts were digested with protease K as in (b),followed by Western analysis with Ab-1. OM lane is the Western analysisof outer membrane fraction. (d) Mitoplasts were digested with protease Kfollowed by Western analysis with Ab-N. (e) Fluorescence intensities ofepitope mapping by Ab-1 and Ab-N. Purified mitochondria or mitoplastswere incubated with Ab-1 or Ab-N, followed by secondary antibodyconjugated with FITC. The fluorescence intensities of the washed pelletswere read by a microplate reader.

FIG. 5. Disruption of mitochondrial transmembrane potential and releaseof cytochrome c by PLS3β in vitro. (a) Purified PLS313 protein (0.1, 0.5or 1.0 μg) was incubated with freshly isolated mouse mitochondria (100μg by protein) and analyzed for Δψ with Rhodamine 123. Calcium (100 or200 μM) was used as positive control to disrupt Δψ. The fluorescenceintensity at 590±35 nm was measured by microplate reader. NC is negativecontrol of mitochondria treated with buffer only. (b) Supernatants ofthe mixtures of PLS3 and mitochondria were analyzed by Western withmonoclonal antibody against cytochrome c. The first two lanes aresupernatant and mitochondrial pellet treated with buffer only forcontrol.

FIG. 6. In vitro phospholipid transfer assays. (a) Phospholipid transferassays with PC, PE and cholesterol. Liposomes containing ¹⁴C-labeledphospholipids were incubated with mouse liver mitochondria along witheither BSA or PLS3 (20 μg) for 20 min. Mitochondria were washed andcounted. Each bar is an average of three experiments. (b) Cardiolipintransfer assays were performed by incubating cardiolipin liposomes,mitochondria and either BSA or PLS3 protein. The washed mitochondriawere then mixed with 30 μM NAO for measurement of fluorescenceintensities at 590 nm. NC: negative control with buffer only. Each baris an average of three experiments.

FIG. 7. Redistribution of cardiolipin at the outer membranes ofmitochondria after UV irradiation and Bcl-B expression. (a) 293 cellswith or without UV irradiation were fixed and stained with variousconcentrations of NAO. Fluorescent intensities of NAO were measured by amicroplate reader at 530±25 nm and 680±30 nm. The curve in 680±30 nm wasconverted to percentage relative to the maximal fluorescence intensityat 35 μM NAO in longitudinal axis. The positions of the shoulders oneach curve are marked by arrowheads. Each point is the average of fourmeasurements. (b) 293-PLS3 cells were analyzed as in (a). (c) 293-PLS3cells were transfected with pcDNA-Bcl-B and subjected to similaranalysis as in (a) to determine the percentage of cardiolipin at theouter membrane of mitochondria. Arrowheads point to the shoulder of thecurves.

FIG. 8. PLS3 is a downstream target of Bcl-B. (a) Synergism of Bcl-B andPLS3 in enhancing apoptosis. 293T cells were transfected with equalamount of control, Bcl-B, PLS3 or both PLS3 and Bcl-B expressionplasmids, and pictures were taken 48 hours after transfection. Theapoptotic cells appear as round and shining circles. (b) Model of Bcl-Band PLS3 interaction and activation of apoptosis in mitochondria.

FIG. 9: (a) Genomic structure of PLS3 and the two alternatively splicedforms. The open boxes are exons, and the solid boxes represent thecoding region. (b) Northern analysis of PLS3 in multiple human tissueblot. The radioactive probes at 2×10⁶ cpm/ml were used forhybridization, and the PLS3 blot was exposed for 24 hours and the actinblot was exposed for 8 hours. The messages of PLS3α and PLS3β wereindicated by arrows.

FIG. 10. Interaction of PLS3 with hBoo and Bcl-2. (a) HEK293 cells weretransfected with pEGFP-hBoo or pEGFP control and IP was performed withPLS3 antibody. The Western blot was probed with EGFP antibody. (b) HeLaand HeLa-Bcl-2 cells were immunoprecipitated with PLS3 antibody, and theWestern blot was probed with Bcl-2 antibody. The positions of the Bcl-2protein are indicated with an arrow. (c) Subcellular fractionation forWestern analysis with PLS3 antibody. Mouse liver were harvested andsubjected to differential centrifugation to obtain nuclei, mitochondria,microsomes and cytoplasm. Equal amounts of protein (20 μg) were loadedinto the gel for separation, and Western analysis was done with PLS3antibody. Same blot was probed with VDAC, tubulin antibody for control.(d) PLS3 is integrated in the mitochondria. Isolated mouse livermitochondria were washed with 0.1 M Na₂CO₃, and the supernatants andpellets were analyzed with PLS3 antibody in Western blot.

FIG. 11. PLS3 is present in mitochondria. Co-localization of EGFP-PLS3with mitochondrial dye MitoTracker Red. HEK293 cells were transfectedwith EGFP-PLS3-α expression vector and stained with MitoTracker Red (100μM). The EGFP-PLS3-β displayed an identical pattern. Control EGFP had adiffuse cytoplasmic pattern.

FIG. 12. Topology of PLS3 in the mitochondria. (a) Six potentialtopologies of PLS3 in mitochondria given that PLS3 has two TM domains.The amino acids of the TM domains were numbered. Two epitopes that wereused to raise antibodies were marked by solid bars (Ab-N and Ab-1). (b)Purified mitochondria were subjected to limited proteinase K digestionfollowed by Western blot analysis with PLS3 Ab-1. (c) Mitochondria weresubjected to limited proteinase K digestion at various temperatures orin a time course from 0-30 minutes. The digested products were analyzedby PLS3 Ab-1. The lower arrows indicate the digested products. The firstlane of temperature study is undigested control. (d, e) Proteinase Kdigestion of mitoplasts. Mitoplasts were digested at variousconcentrations of proteinase K. Western analysis was performed with Ab-1(d) and Ab-N (e). The positions of PLS3 and its degradation products aremarked. The molecular weight standards are indicated in left. (f)Epitope mapping of PLS3 with Ab-1 and Ab-N. Mitochondria or mitoplastswere incubated with Ab-1 or Ab-N followed by a secondary antibodyconjugated with FITC. PK 0 or 1.0: No proteinase K digestion or digestedwith 1.0 μg before incubation with PLS3 antibody. The fluorescence ofthe washed pellets was next measured. NC: negative control by incubatingmitochondria with secondary antibody only.

FIG. 13. Stably-transfected cell lines expressing wild-type PLS3 ormutant PLS3(F258V). (a) G418-resistant clones of HEK293 cellstransfected with pcDNA, pcDNA-PLS3 or pcDNA-PLS3(F258V) were harvested,and whole cell lysates analyzed by Western blotting with anti-PLS3antibody. (b) 293-vector, 293-PLS3 and 293-PLS3(F258V) cells werefractionated, and mitochondrial (M) and cytoplasmic (C) fractions wereanalyzed by Western blotting with antibodies against PLS3, VDAC andtubulin. (c) Growth curves of the 293-vector, 293-PLS3 and293-PLS3(F258V) cells under normal growth conditions. Cells were platedon day 0, and counted at days 1, 2 by trypan blue exclusion. (d) Growthcurves in the presence of Na₃N at three different concentrations.

FIG. 14. Overexpression of mutant PLS3 reduces mitochondrial mass,potential, and cytochrome c and CL content. A. 293-vector, 293-PLS3, and293-PLS3(F258V) cells were incubated with JC-1 dye as indicated andgreen (left panel) and red (right panel) fluorescence were determined byflow cytometry. B. Flow cytometry curves for 293-vector, 293-PLS3, and293-PLS3(F258V) cells stained with Rhodamine 123. The left panel showsthe decrease of mitochondrial potential by treatment of 293-vector cellswith 20 AM antimycin A for 6 h. The right panel shows the mitochondrialpotential determined by Rhodamine 123. C. Western blotting of whole celllysates from 293-vector (lane 1), 293-PLS3 (lane 2), and 293-PLS3(F258V)cells (lane 3). The blot was re-probed with antibodies to VDAC andtubulin for controls. D. 293-vector, 293-PLS3, and 293-PLS3(F258V) cellswere stained with 10-N-nonyl-3,6-bis(dimethylamino) acridine orange(NAO), and fluorescence intensities at 570 nm were measured. Error bars,SDs from five independent measurements. E. Quantitative PCR analysis ofmitochondrial NADH dehydrogenase in 400 ng of whole cell genomic DNA.Curves 1-3, different concentrations of standards; other curves,quantification of 293-vector, 293-PLS3, and 293-PLS3(F258V) intriplicates. The crossing points (in cycle numbers) of the three cellsare indicated.

FIG. 15. Analysis of mitochondrial respiration. (a) The ATPconcentrations of the HEK293-vector, HEK293-PLS3, and HEK293-PLS3(F258V)cells were measured by luciferase assays. The results are the averagesof five experiments. (b) The oxygen consumption of the cells wasmeasured with oxygen electrode by dissolved oxygen meter as described inmethods. Mitochondria (50 μg) were isolated from HEK293-vector,HEK293-PLS3 and HEK293-PLS3(F258V) cells and placed in the chamber ofMitocell. The state 4 respiration was induced with succinate at a finalconcentration of 7 mM, and oxygen concentrations in the chamber weremonitored for 2 min. ADP was then added into the same chamber to a finalconcentration of 150 μM to measure oxygen consumption in state 3respiration for another 6 min. (c) Oxygen consumption in states 3 and 4respiration. The slopes of each curve in b were determined and theoxygen consumption rates (pmole/min/μg mitochondrial protein) werecalculated. The results are the averages of 3 independent experiments.

FIG. 16. Electron microscopic examination of the HEK293-vector,HEK293-PLS3, and HEK293-PLS3(F258V) cells. All are 33,047×foldmagnification.

FIG. 17. UV irradiation of cells overexpressing PLS3 or PLS3(F258V)mutant. (a) HeLa, HeLa-vector, HeLa-PLS3 and HeLa-PLS3(F258V) cells wereirradiated with UV at 4 J/m²/sec for 2 min followed by staining with MTTas described in methods. Shown are the averages of 5 independentmeasurements. (b) Cells were irradiated with UV as in (a), and thepercentages of apoptosis was analyzed by annexin V-PE binding asdescribed in methods. The percentages of annexin V-PE positive cells areindicated. (c) Cells were treated with or without UV and analyzed byJC-1. JC-1 is shown on the left and JC-1 red is shown on the right.

FIG. 18. PLS3 regulates CL content and apoptotic translocation. A.Mitochondria were fractionated into IM and OM, and the percentages ofenzyme activities of MAO and MDH in the mitochondrial OM and IMfractions were determined. B. 293-vector cells were labeled with[³²P]Pi, UV-treated, and then mitochondrial IM and OM were isolated andlipids extracted for TLC analysis. Migration of phosphatidylcholine(PC), phosphatidylethanolamine (PE), and CL was established bynonradioactive standards followed by iodine staining (standards). C.³²P-labeled lipids were analyzed from equal amounts of mitochondria from293-PLS3 or 293-PLS3(F258V) cells with or without UV irradiation as inB. D. Shown are percentages of CL present in the OM of unirradiated(open bars) and UV-treated (shaded bars) 293-vector, 293-PLS3, and293-PLS3(F258V) cells, derived from the ratio of OM to the sum of OM andIM. Error bars, SDs from three experiments.

FIG. 19. Effects of PLS3 on tBid-induced cytochrome c and SMAC release.A. Mitochondria (60 μg) isolated from HeLa-vector (lanes 1 and 4),HeLa-PLS3 (lanes 2 and 5), and HeLa-PLS3(F258V) cells (lanes 3 and 6)were incubated with buffer (lanes 1-3) or recombinant tBid (0.6 μg;lanes 4-6) for 20 min at 37° C. The supernatants and pellets weresubjected to Western analysis for cytochrome c, SMAC, and VDAC. B. Thebands of cytochrome c and SMAC in A were quantified by densitometry andthe percentages of tBid-induced mitochondrial release are shown.

FIG. 20. (a) PLS3 is phosphorylated at threonine. HEK293 or HEK293-PLS3cells were treated with or without UV. Cell lysates wereimmunoprecipitated with PLS3 antibody. Western blot analysis was thenperformed with PS, PT, PY, PLS3 and PKC-δ antibodies. (b) UV irradiationinduces apoptosis requires PKC-δ. HEK293 cells were treated with UV inthe presence or absence of PKC-δ inhibitor, rottlerin (10 μM). Cellswere harvested 4 hours later and fractionated to isolate mitochondriaand cytosols. Fractions were analyzed with Western blot with cytochromec, VDAC, tubulin antibodies. In the bottom two panels, the mitochondrialfractions were further washed with mitochondrial isolation buffer toremove rottlerin. The washed mitochondria were then incubated with 100nM PMA to activate PKC-δ to determine if they were translocated to themitochondria. After 20 minutes incubation, the mitochondria wereseparated from the supernatants. The supernatants and pellets wereanalyzed for cytochrome c.

FIG. 21. Enzyme kinetics of PKC-δ phosphorylating PLS3. a, in vitrophosphorylation of PLS3 with recombinant PKC-δ in a time course from 0to 25 min. Recombinant PLS3 protein (1 μg) and [γ-32P]ATP were used assubstrates, and recombinant PKC-δ was used as kinase for each in vitrophosphorylation reaction. b, kinetics study of PKC-δ toward PLS3 wasperformed using various concentrations of PLS3 and equal amount of PKC-δfor each reaction. The reaction was stopped after 20 min of incubationand analyzed by gel electrophoresis. The radioactivity of each PLS3protein was determined by density analysis and plotted against theconcentrations of the PLS3 protein. The plot in c is thedouble-reciprocal plot derived from the results to calculate the k_(m)value.

FIG. 22. Overexpression of PLS3 converts PMA to a cell death agonist.HeLa-control, HeLa-PLS3, and HeLa-PLS3(F258V) cells were incubated withDMSO (black), 200 nM PMA (green) or the combination of 200 nM PMA and200 nM Go6976 (red) for 2 hours. Cells were then fixed with ethanol forTUNEL assays to quantify apoptosis.

FIG. 23. Overexpression of mitochondrial targeted PKC-δ. (a) AnnexinV-PE study of the apoptosis was performed in the HeLa-control, HeLa-PLS3and HeLa-PLS3(F258V) transfected with the mitochondrial targeted PKC-δconstruct or control vector. The percentages of apoptosis wereindicated. (b) immunofluorescent staining was performed in thevector-transfected control HeLa cells with PKC-δ antibody forcolocalization with MitoTracker Red. (c) HeLa cells were transfectedwith expression vector of the mitochondrial targeted PKC-δ. Similarstaining was performed with PKC-δ and MitoTracker Red. PKC-δ antibodystaining (left panels); MitoTracker Red (middle panels); overlay (rightpanels).

DETAILED DESCRIPTION OF THE INVENTION

The present invention will be best understood by reference to thedrawings and detailed description that follows. The following moredetailed description of the gene, and methods of inducing apoptosis andcreating apoptosis resistance using related genes of the presentinvention, as represented in FIGS. 1 through 23, is not intended tolimit the scope of the invention, as claimed, but is merelyrepresentative of presently preferred embodiments of the invention.

As known to one of ordinary skill in the art, both nucleic acidsequences and amino acid sequences may be varied within the scope of theinvention. More specifically, changes or mutations to either amino acidor nucleic acid sequences that are conservative are included within thescope of the invention. In the case of nucleic acids, such conservativesubstitutions may produce a protein having a sequence 85%, 90%, 95%, oreven greater than 95% identical to the sequences disclosed herein andmaintaining the function of the disclosed proteins. Such nucleic acidsequences and amino acid sequences are considered within the scope ofthe instant invention. In addition, mutations or deletions that haveminor or no consequence on the function of the nucleic acid or aminoacid are considered within the scope of the invention. Similarly,nucleic acids composed of naturally-occurring nucleotides, sugars andinternucleotide (or “backbone”) linkages, as well as oligonucleotideshaving modified nucleotides, sugars, or backbone linkages, as well asoligonucleotides having mixed natural and modified nucleotides, sugars,and backbones or other non-naturally occurring portions that havesimilar function to naturally-occurring compounds are considered withinthe scope of the invention.

I. Phospholipid Scramblase 3 is a Downstream Effector of Cell DeathRegulator Bcl-B

Bcl-B is the human orthologue of mouse BOO/Diva, a member of Bcl-2family similar to avian NR13. Avian NR13 was originally identified as av-src-activated gene (Gillet et al. 1995), and overexpression of NR13protein protected Baf-3 cells from IL-3 withdrawal-induced apoptosis(Mangeney et al. 1996). NR13 plays a major role in regulation of chickenbursal apoptosis. NR13 protected a bursa-derived cell line, DT40, fromserum-deprivation-induced apoptosis, and the level of NR13 correlatedwith bursal cell survival in vivo. Using a bursal transplantation model,it was demonstrated that one physiological function of NR13 is toprotect the bursal stem cells from programmed elimination (Lee et al.1999).

Searching for mammalian homologues of avian NR13, two groups identifiedmouse BOO/Diva through EST database analysis (Inohara et al. 1998; Songet al. 1999). It has a unique tissue distribution, expressed in allembryonic tissues but only in adult ovary. Protection from or promotionof cell death by BOO/Diva was cell-type dependent. BOO/Diva interactedwith Apaf-1; however, the physiological significance of this interactionis unclear (Inohara et al. 1998; Song et al. 1999).

Bcl-B is highly conserved between mouse and human, and its anti- orpro-apoptotic effect is significantly weaker compared with Bcl-2 orBcl-xL. Bcl-B interacts with Bcl-2, Bcl-xL and Bax, but not Bak (Ke etal. 2001). Bcl-B is not completely localized in mitochondria, buttranslocates to mitochondria upon induction of cell death bymicrotubule-interfering agents. Bcl-B gene was mapped in chromosome15q21, and deletion was identified in some primary cervical cancers (Leeet al. 2001).

The biochemical functions of Bcl-2 family in mitochondria are far fromclear. The crystal structure of Bcl-xL (Muchmore et al. 1996) suggestedthat Bcl-2 family members could form channels, and regulate themitochondrial transmembrane potential (Δψ) and the release of cytochromec upon pro-apoptotic stimulation (Shimizu et al. 1999; Vander Heiden andThompson 1999; Shimizu et al. 2000; Shimizu and Tsujimoto 2000). Theregulation of mitochondrial transmembrane potential (Δψ) is mediatedthrough a transmembrane permeability pore complex (Shimizu et al. 1999).This multiprotein complex contains many components, includingvoltage-dependent anion channel (VDAC) and peripheral benzodiazepinereceptor (PBR) on the outer membrane of mitochondria, adenine nucleotidetranslocator (ANT) on the inner membrane, and a matrix proteincyclophilin D (CphD).

Shimizu et al. used liposomes carrying VDAC to show that Bax and Bakaccelerated the opening of VDAC, while Bcl-xL closed VDAC through directinteraction with VDAC. Using a BH4 domain peptide of Bcl-xL, theyeffectively prevented apoptotic cell death induced by etoposidetreatment, indicating that BH4 domain of Bcl-xL was operative in thisfunction (Shimizu et al. 2000). On the other hand, the BH3-only Bcl-2family members induce release of cytochrome c without Δψ loss, and donot directly modulate VDAC activity (Shimizu and Tsujimoto 2000). Theseresults demonstrated two different functions of the mitochondrialtransmembrane pore complex, i.e. maintaining mitochondrial transmembranepotential (Δψ) and serving as a transmembrane protein channel, and theyare regulated through different domains of Bcl-2 family members.

Cardiolipin has recently drawn attention as a mitochondrial phospholipidinvolved in apoptosis. This dimeric phospholipid contains twophosphatidyl molecules linked by a glycerol moiety, thus in total fourfatty acyl chains and two phosphate groups (Hoch 1992). Cardiolipin ismainly located at the inner membranes of mitochondria (Krebs et al.1979) and is required for oxidative phosphorylation and high energyelectron transport (Jiang et al. 2000; Schlame et al. 2000). Cardiolipininteracts with cytochrome c, and peroxidation of cardiolipin dissociatescytochrome c from cardiolipin (Shidoji et al. 1999; Nomura et al. 2000).

Studies of cardiolipin were carried out with a cardiolipin-specificfluorescence dye NAO, nonyl-acridine orange (Garcia Fernandez et al.2000). NAO has a fluorescence emission at 625 nm with 2:1 stoichiometricinteraction to cardiolipin, and at 525 nm with 1:1 interaction.Measuring the amount of cardiolipin can be done with NAO by flowcytometry analysis (Garcia Fernandez et al. 2000). When cells becomeapoptotic, NAO binding decreases secondary to peroxidation ofcardiolipin, a process that contributes to the release of cytochrome c(Shidoji et al. 1999). Another function of cardiolipin was recentlyidentified as the mitochondrial target for tBid, which is cleaved fromBid upon apoptotic stimulation and induces activation of caspase 9 alongwith cytochrome c and Apaf-1 (Lutter et al. 2000).

The identification of a mitochondrial phospholipid scramblase 3 (PLS3)which interacts with Bcl-B is reported herein. It was found that PLS3has a unique topology of crossing both outer and inner membranes ofmitochondria, and PLS3 moves cardiolipin from the inner to the outermembranes of mitochondria. Overexpression of PLS3 significantly enhancedthe pro-apoptotic effect of Bcl-B, and a PLS3 mutant preventsBcl-B-induced cell death. These results indicate that PLS3 is adownstream effector of Bcl-B.

II. Phospholipid Scramblase 3 is a Member of the Scramblase Family thatis Present in the Mitochondria

As previously noted, phospholipid scramblases are a family of enzymesresponsible for bidirectional movement of phospholipids between twocompartments (Bevers et al., 1999; Sims and Wiedmer, 2001). Four membersof PLS family have been identified in the GenBank database (Wiedmer etal., 2000). PLS1 is the best-studied member and localizes in the plasmamembrane. The remaining three members of the family are essentiallyuncharacterized.

During apoptosis, phosphatidylserine (PS) is translocated from the innerleaflet to the outer leaflet of the plasma membrane as a phagocytoticsignal for macrophages (Fadok et al., 1992; Martin et al., 1995; Brattonet al., 1997; Zhao et al., 1998; Fadok et al., 2000; Fadok et al.,2001). It was suspected that the activity of PLS1 is related with PSflipping. However, cells from homozygous PLS1-deficient mice still havePS flipping in apoptosis presumably through complementation by otherphospholipid translocase activities (Zhou et al., 2002). PLS1 can bephosphorylated at threonine and tyrosine by PKC-δ and c-abl kinasesrespectively (Frasch et al., 2000; Sun et al., 2001). However, itremains to be confirmed that the phosphorylation directly results inPLS1 activation (Zhao et al., 1998).

It is disclosed herein that another member of the PLS family, PLS3, islocalized in the mitochondria. In mitochondria, PLS3 interacts withmember of the Bcl-2 family, the human Boo/Diva (hBoo) and Bcl-2. HumanBoo can either enhance or inhibit apoptosis similar to the mouse gene(Inohara et al., 1998; Song et al., 1999; Aouacheria et al., 2001; Ke etal., 2001; Lee, 2001). Mutations in hBoo were identified in humancervical cancer (Lee, 2001), but the significance and the biologicalfunction of hBoo remain unknown. Recently, the Boo/Diva deficient micewere generated by homologous recombination, but no obvious phenotype wasobserved (Russell et al., 2002).

Results and Discussion:

Cloning of PLS3 by interaction with hBoo.

To understand the function of hBoo, its interacting protein was soughtby yeast two-hybrid screening of a human liver cDNA library. Among the17 clones that interacted specifically with hBoo but not with thecontrol bait lamin C, three were identified as cDNA encoding amino acids103-277 of phospholipid scramblase 3 (Wiedmer et al., 2000). Thefull-length cDNA clone was then identified (AW239215; accession no.6571605, SEQ ID NO: 11) by searching the human EST database. The wholeAW239215 clone was sequenced, and it was noted that it is a differentisoform compared with the cDNA cloned from the yeast two-hybrid screen.The AW239215 clone contained 1722 nucleotides between the 5′ end cloningsite and the poly A tail. Nucleotides 1017-1596 were missing in the cDNAclone previously obtained. The translated proteins differ only in thelast 17 amino acids. The shorter form was designated PLS3-α and thelonger form (AW239215) as PLS3-β. Using the Ensembl human genomedatabase to look for the genomic structure of PLS3, it was found thatPLS3 is located in human chromosome 17 at the telomeric side of p53 geneabout 0.4 Mb in distance (Ensembl gene ID: ENSG00000174289), and themouse orthologue is located at the corresponding mouse chromosome 11.The genomic structure of human PLS3 contains 8 exons and the last exonis alternatively spliced to generate PLS3α and PLS3β (FIG. 9 a). Bothsplice junctions contain the required AG sequence right before thesplice sites (the GT-AG rule). The two alternatively spliced messagesare consistent with the two signals (2.4 and 1.8 kilonucleotides insize) in the northern blot (FIG. 9 b). Blast search identified mouse,Drosophila and C. elegans homologues, indicating that this gene ishighly conserved in evolution (FIG. 1).

The in vivo interaction of PLS3 and hBoo was confirmed byimmunoprecipitation (IP). HEK293 cells were transiently transfected withpEGFP-hBoo expression vector, and lysates were immunoprecipitated withaffinity-purified PLS3 antibody. FIG. 10 a shows that hBoo specificallyco-precipitated with PLS3; while the control IP from HEK293 cellstransfected with pEGFP vector failed to bring down EGFP by PLS3antibody. This result indicates a physical interaction between PLS3 andhBoo. Since hBoo is a member of the Bcl-2 family, it was also studiedwhether PLS3 interacts with other members of the Bcl-2 family. Theinteraction with Bcl-2 was examined in both HeLa cells and HeLa cellsoverexpressing Bcl-2. The endogenous PLS3 from HeLa cellsco-immunoprecipitated with Bcl-2, and more Bcl-2 was present in the PLS3immunoprecipitates from HeLa-Bcl-2 cells (FIG. 10 b). The control IPswith preimmune serum were negative. Similar study with Bcl-xL antibodywas negative despite multiple attempts (data not shown), indicating thatPLS3 binds Bcl-2 but not Bcl-xL.

Both PLS3-α and PLS3-β localize to mitochondria.

The interaction of PLS3 with members of the Bcl-2 family suggests thatPLS3 may have a different localization from PLS1. Because our antibodycould not detect native PLS protein by immunofluorescence staining, thesubcellular localization of PLS3-α and PLS3-β was examined fused withEGFP at their N-terminus. Both EGFP-PLS3-α and EGFP-PLS3-β had a similargranular pattern. Cells were stained with MitoTracker Red, amitochondrial dye, for co-localization. EGFP-PLS3 co-localized withMitoTracker Red (FIG. 11 a), and cells transfected with control EGFPplasmid has a diffuse cytoplasmic pattern (FIG. 11 b). This indicatesthat both PLS3-α and PLS3-β are in the mitochondria. Subcellularfractionation of mouse liver was then performed, and a Western blotconfirmed that endogenous PLS3-α is predominantly present inmitochondria (FIG. 10 c). The isolated mouse liver mitochondria werewashed with Na₂CO₃ and confirmed that PLS3 can not be washed off with0.1 M Na₂CO₃, and therefore is integrated in the mitochondria (FIG. 10d). No mitochondrial targeting sequence could be identified at theN-terminus of PLS3.

Proteins with mitochondrial targeting sequence are imported by theTIM/TOM complex into the matrix (Bauer et al., 2000). Severalmitochondrial proteins, such as Bcl-2, VDAC, also lack any detectablemitochondrial targeting sequence since they do not depend on TIM/TOMcomplex for mitochondrial integration.

Topology of PLS3 in the Mitochondria

The topology of PLS3 in mitochondria was next investigated. Computeranalysis (http://www.ch.embnet.org/software/TMPRED_form.html) of PLS3αpredicted two transmembrane (TM) domains at amino acids 50-70 and266-286, in contrast to one TM domain for PLS1. Given its two putativeTM domains, PLS3 has six potential topologies with respect to themitochondrial IM and OM. PLS3 could cross the OM twice (topologies 1,6), the IM twice (topologies 2, 5), or both IM and OM once (topologies3, 4) (FIG. 12 a). Two different approaches were used to study thetopology: proteinase K digestion and antibody epitope mapping. (Donzeauet al., 2000). It was anticipated that the proteinase K digestion of theintact mitochondria would generate a very small size PLS3 fragmentrecognized by Ab-N in topology 1, a slightly smaller fragment of PLS3recognized by both Ab-1 and Ab-N in topologies 3, 4, and 6, and no PLS3digestion in topologies 2 and 5 because they are buried inside ofmitochondria.

Due to the concern that overexpressed protein may have aberranttopology, mitochondria isolated from mouse liver were used, which haveabundant native PLS3 protein (Wiedmer et al., 2000). By incubatingmitochondria in HEPES-mannitol buffer to preserve the mitochondrial OM,a dose-dependent proteinase K digestion was performed to cleave theportion that is outside of mitochondria, and analyzed the digestedmitochondria with Ab-N and Ab-1 antibodies. Western analysis of thedigested mitochondria revealed that the 30% D PLS3 protein was digestedto a smaller fragment migrating at 25 kD (lower arrow, FIG. 12 b).Similar results were obtained in a limited proteinase K digestion from 5min to 30 min, or 4° C. to 25° C. (FIG. 12 c). These results indicatethat the PLS3 protein in intact mitochondria is accessible to proteinaseK, and that a small portion of PLS3 is present outside of mitochondria.Topologies 1, 2, 5 could thus be eliminated.

Next, purified mitochondria were incubated in a low concentration HEPESbuffer to generate mitoplasts by osmotic swelling and disruption of theOM. Using MAO and MDH, it was shown that this mitoplast preparationcontained 76% of total MDH activity and 20% of total MAO activity, andthe OM fraction contained 24% of MDH and 80% of MAO activity (Ragan,1995). Although mitoplasts still contained about 20% of the residual OM(likely sticking to mitoplasts at the junctional zone), proteinase Kcould still have access to the intermembranous space through the holesgenerated by osmotic swelling, and is capable of digesting proteins atthe intermembranous space (Donzeau et al., 2000). It was anticipatedthat digestion of the portion in the intermembranous space wouldgenerate a fragment of 70 amino acids (the N-terminal 50 amino acidsplus the 20 amino acids of the TM domain) only recognized by Ab-N, butnot Ab-1, in topology 3. In topologies 4 and 6, both epitopes recognizedby Ab-1 and Ab-N would be digested, resulting in no fragments in Westernblot.

Western analysis of proteinase K-treated mitoplasts revealed furthercleavage of the 25 kD product to smaller fragments at sizes of 15-25 kD(FIG. 12 d). The undigested OM fraction failed to show any PLS3 protein(FIG. 12 d far right lane). These two findings eliminated topology 6, inwhich PLS3 should only be present in the OM. In Western analysis withAb-N, a 9 kD band was detected by Ab-N at the highest concentration ofproteinase K digestion (FIG. 12 e). This is compatible with topology 3with only the first 70 amino acids remaining after digestion of theintermembranous portion of PLS3. It was thus concluded that topology 3is the actual orientation of PLS3 in mitochondria, with the N-terminusin the matrix and the C-terminus on the outer surface of mitochondria.

Epitope mapping was then performed to study the localization of theepitopes recognized by the anti-PLS3 antibodies discussed above. Asshown in FIG. 12 f, intact mitochondria failed to be recognized byeither Ab-1 or Ab-N, suggesting that neither epitope is outside of themitochondria. Mitoplasts were recognized by Ab-1, but not by Ab-N (FIG.12 f), indicating that the epitope between the two TM domains recognizedby Ab-1 resides in the intermembranous space, and that the N-terminusepitope recognized by Ab-N is inside the mitoplasts. Limited proteinaseK digestion of the mitoplasts decreased Ab-1 recognition, confirmingthat the epitope of Ab-1 is outside of mitoplasts (FIG. 12 f). Theseresults are also consistent with the topology 3 (FIG. 12 a). Therefore,both proteinase digestion and epitope mapping support that PLS3 proteincrosses both mitochondrial IM and OM with the N-terminus in the matrixand the C-terminus outside of mitochondria. This type of mitochondrialtopology has been described in Tim23, a subunit of the mitochondrialpre-protein translocase complex (Donzeau et al., 2000). This pre-proteintranslocase complex is responsible for translocating the pre-proteinsthat are synthesized in the cytoplasm into mitochondria. Without beinglimited to any one theory, it is thought that PLS3 could be responsiblefor moving phospholipids into or out of mitochondria. Unfortunately,very few studies were done regarding the movement of phospholipids intomitochondria, although the bioactive lipids are well known to play rolesin the signal transduction processes. It is not clear whether theytranslocate to mitochondria.

Another possible function of PLS3 is to move phospholipids between theinner and outer membranes of the mitochondria based on its topology ofexpanding through both IM and OM. The phospholipid composition betweenthe inner and outer membranes of mitochondria is different (Parsons,1967; Parsons and Yano, 1967). The most dramatic difference inphospholipids is the amount of cardiolipin, a mitochondrion-specificphospholipid. Cardiolipin is predominantly present in the inner membraneof mitochondria and plays a role in oxidative phosphorylation and ATPproduction. Without being limited to any one theory, maintaining thecardiolipin homeostasis appears to be important for the normalmitochondrial functions. It remains to be determined whether PLS3 playsany role in mitochondrial phospholipid homeostasis.

III. Phospholipid Scramblase 3 Controls Mitochondrial Structure,Function, and Apoptopic Response

Regulation of programmed cell death is central to development andneoplastic transformation. Dysregulation of apoptotic mediators resultsin tumor clonal evolution and resistance to chemotherapy (Ionov et al.,2000; Reed, 1999). Morphologically, apoptosis is characterized first bymitochondrial disintegration, followed by nuclear fragmentation,chromatin condensation and cytoplasmic membrane blebbing (Cory andAdams, 1998; Kroemer and Reed, 2000).

Mitochondria are the integrators of many apoptotic pathways. Severalpro-apoptotic regulators, including Bax, Bad, Bid, Bim, p53, JNK, PKC-δ,nuclear receptor TR3 (Brenner and Kroemer, 2000), and the Peutz-Jeghergene product LKB1 (Karuman et al., 2001), translocate to themitochondria during apoptosis. Among these factors, Bid is cleaved atthe N-terminus to become tBid by activated caspase 8 beforemitochondrial targeting (Luo et al., 1998). This targeting of tBid ismediated by N-myristoylation (Zha et al., 2000) and interaction withcardiolipin (Lutter et al., 2000). Activated tBid inducesoligomerization of Bax and Bak to form cytochrome c channels duringapoptosis (Korsmeyer et al., 2000; Wei et al., 2000; Wei et al., 2001).Without the presence of Bax or Bak, cytochrome c release or apoptosis isresistant to tBid (Wei et al., 2001).

During apoptosis, phosphatidylserine (PS) is translocated from the innerto the outer leaflet of the plasma membrane (Bratton et al., 1997; Fadoket al., 1992; Martin et al., 1995). Macrophages recognize PS in theouter membrane, and engulf apoptotic cells by phagocytosis (Fadok etal., 2000; Fadok et al., 2001). However, the regulation of the enzymesresponsible for PS translocation remains elusive.

Phospholipid scramblases (PLS) are enzymes responsible forbi-directional movement of phospholipids (Bevers et al., 1999), and fourPLS family members have been identified (Wiedmer et al., 2000). PLS1 islocated in the plasma membrane and responsible for translocation ofphospholipids between the inner and outer leaflets (Zhou et al., 1997).Although apoptotic PS translocation was unaltered in PLS1-deficient mice(Zhou et al., 2002), the role of PLS1 in apoptosis remains unclear giventhe presence of additional enzymes associated with plasma membranephospholipid translocation such as aminophospholipid translocase (Beverset al., 1999; Williamson et al., 2001; Zhao et al., 1998). PLS familymembers contain a conserved calcium-binding motif, and Zhou et al (Zhouet al., 1998) found that mutation of residues in this region of PLS1completely eliminated enzymatic activity. PLS1 is phosphorylated atThr-161 by PKC-δ, which translocates to the plasma membrane duringapoptosis (Frasch et al., 2000); in addition, PLS1 is phosphorylated atTyr-69/74 by c-abl kinase (Sun et al., 2001). The role of calciumbinding and these various phosphorylation events in PLS 1 activation andregulation, however, remain to be determined.

A newly identified member of the scramblase family, designated PLS3, islocalized to the mitochondrial rather than plasma membrane (Liu et al.,2003). However, little is known regarding the physiologic function ofPLS3 in mitochondria. It is shown herein that PLS3, like PLS1, isphosphorylated by PKC-δ (Liu et al., 2003). A mitochondrial targetedPKC-δ dramatically enhanced susceptibility to apoptosis in cellsoverexpressing PLS3, suggesting that PLS3 is the direct mitochondrialeffector of PKC-δ-induced apoptosis (Liu et al., 2003). It is reportedherein that targeting PLS3 disrupts both mitochondrial structure andfunction, including translocation of cardiolipin from the mitochondrialinner membrane (IM) to the outer membrane (OM) during apoptosis.

Members of the protein kinase C (PKC) family of kinases play diverseroles in cell signaling, proliferation, apoptosis, and other cellularprocesses. PKC-δ is particularly associated with apoptosis. PKC-δ ispresent in the cytoplasm, and translocates to various organelles,including the nucleus, mitochondria and the plasma membrane uponapoptotic stimulation. One substrate of PKC-δ is phospholipid scramblase1 (PLS1) in the plasma membrane. However, the consequence of PLS1phosphorylation and how PLS1 contributes to apoptosis remain elusive.PKC-δ is activated during apoptosis by caspase-mediated cleavage tobecome catalytically active. This catalytic active fragment of PKC-δtranslocates to the mitochondria to induce apoptotic events. Inhibitionof PKC-δ blocks the disruption of the mitochondrial transmembranepotential. These results indicate that PKC-δ plays an important role intriggering apoptosis through a mitochondrion-dependent pathway. However,the substrate of PKC-δ in the mitochondria has previously beenunidentified.

The function of phospholipid scramblase (PLS) is to translocatephospholipids bidirectionally between two compartments. PLS1translocates phospholipids between the inner and outer leaflets of theplasma membrane, which is asymmetric in lipid composition. Althoughphosphatidylserine (PS) is translocated to the outer leaflet duringapoptosis, it is still unclear whether PLS1 is responsible for thistranslocation. PLS3, which is present in the mitochondria, is discussedin detail herein. Overexpression of PLS3 in the HEK293 cells enhancedapoptosis induced by UV irradiation, and blocking PLS3 with a dominantnegative mutant of PLS3 suppressed UV and etoposide-induced apoptosis(Dai, submitted). It is reported herein that PLS3 interacts with PKC-8,and is phosphorylated by PKC-δ with a very high affinity in vitro.

Cells overexpressing PLS3 became apoptotic upon treatment with phorbolester and upon expression of mitochondrial targeted PKC-δ. These resultsindicate that PLS3 is the downstream target of PKC-δ.

EXAMPLES

I. Phospholipid Scramblase 3 is a Downstream Effector of Cell DeathRegulator Bcl-B

Cloning of PLS3 and Identification of Two Different Forms ofTranscripts.

In order to understand the functions of Bcl-B, a yeast two-hybrid screenwas performed to identify proteins that interact with Bcl-B. Since Bcl-Bis most abundant in human liver (Lee et al. 2001), a yeast human liverMatchMaker cDNA library (Clontech, Palo Alto, Calif.) was used for thisscreening. After initial screening, seventeen true positive clones wereidentified. Three clones were identical to human phospholipid scramblase3 (Wiedmer et al. 2000). Since the clone obtained lacked 5′ sequence,the full-length cDNA clone was identified (AW239215; GI:6571605, SEQ IDNO: 11) by searching the human EST database. The AW239215 clone wassequenced and it was noted that it is an alternatively spliced formcompared with the cDNA cloned. The AW239215 clone contained 1722nucleotides between the Eco RI cloning site and the poly A tail.Nucleotides 1017-1596 were missing in the cDNA clone obtained. Thetranslated proteins differ only in the last 15 amino acids (FIG. 1). Theshorter form was named PLS3α (SEQ ID NO: 2) and the longer form(AW239215) as PLS3β (SEQ ID NO: 4). The hydrophobicity of each wasanalyzed to search for transmembrane domains and found that PLS3αcontains two transmembrane helices at amino acids 51-72 and 270-286.PLS3β contains one good candidate transmembrane helix at amino acids51-72 and a weak hydrophobic helix at amino acids 262-282. Homologousproteins were identified in mouse, Drosophila and C. elegans, indicatingthat this gene is highly conserved in evolution. See FIG. 1. The codingregion of PLS3 is complementary to the cDNA sequence of humannon-receptor tyrosine kinase TNK1 (AF097738) at the 3′ end untranslatedregion. TNK1 is closely linked with the human tumor suppressor gene p53and mapped to human chromosome 17p13 (Hoehn et al. 1996).

The in vivo interaction of PLS3 and Bcl-B was confirmed byimmunoprecipitation (IP). 293T cells were transiently transfected withpEGFP-Bcl-B and pcDNA-PLS3α expression vectors, and harvested forimmunoprecipitation with affinity-purified PLS3 antibody. Westernanalysis was then performed with GFP antibody, which revealed thatEGFP-Bcl-B was present in PLS3 immunoprecipitates (FIG. 2 a). Thisresult demonstrated the physical interaction of PLS3 and Bcl-B proteinsin cells.

Tissue Distribution of PLS3α and PLS3β

Using a Human 12-Lane Multiple Tissue Northern (MTN) blot (Clontech,Palo Alto, Calif.), northern analysis was performed to study human PLS3expression. It was found that PLS3 was expressed in two forms aspredicted from our identification of two alternative splice forms. PLS3αwas a 1.8 K message that was abundantly expressed in human spleen,kidney, liver, placenta, and less abundantly in brain, heart, andperipheral blood leukocytes. PLS3β was a 2.4 K message highly expressedin skeletal muscle, heart, and small amounts in brain. See FIG. 2 b. Ithas been have previously reported that Bcl-B was expressed in humanliver, kidney and ovary (Lee et al. 2001). This finding indicates thatPLS3 has other physiological functions or modulators independent ofBcl-B.

PLS3α and PLS3β were both localized in mitochondria, and an N-terminaldeletion disrupted mitochondrial transmembrane potential.

In order to investigate the subcellular localization of PLS3, PLS3α andPLS3 were fused with green fluorescent protein (GFP). The mitochondriaof the transfected cells were stained with MitoTracker Red dye forco-localization. GFP fusion proteins of both PLS3α and PLS3β hadidentical patterns, and both were co-localized with MitoTracker dye. SeeFIG. 3 a. This indicates that both PLS3α and PLS3β are in mitochondria.To ensure that GFP has no effect on localization, subcellularfractionation of mouse liver and cells transfected with pcDNA-PLS3α wereperformed for Western analysis. PLS3α was predominantly present inmitochondria, with a small amount in nuclei and microsomes, and was notdetectable in cytosol. See FIG. 3 c.

The accumulation of MitoTracker dye in mitochondria of PLS3α and PLS3βtransfected cells indicated that they still had intact mitochondrialtransmembrane potential. They also remained viable after G418 selection.A GFP fusion protein was made with an N-terminal deletion mutant,ΔPLS3α, which lacks the first 100 amino acids. Cells with GFP-ΔPLS3αexpression were not labeled with MitoTracker Red, indicating that theycould not accumulate MitoTracker dye, a process dependent on Δψ. SeeFIG. 3 b. They eventually died after 7 days. This indicated thatoverexpression of GFP-ΔPLS3α disrupted mitochondrial Δψ and eventuallycaused apoptotic cell death, suggesting that the first 100 amino acidsplayed a regulatory role in PLS3 functions.

Determination of PLS3 Topology

Since mitochondria have inner and outer membranes separating theintermembranous space and matrix, the topology of PLS3 in mitochondriawas investigated to facilitate studying the function of PLS3. Rat liverwas used to study the native PLS3α protein with the concern thatoverexpressed protein may not be localized in the appropriate position.By computer prediction, there are two putative TM domains (amino acids50˜70 and 266˜286) in PLS3α protein, the isoform in liver, and thereforesix potential topologies of PLS3 regarding its location at the inner andouter membranes of mitochondria. See FIG. 4 a. PLS3 could be crossingthe inner membrane twice (topology 1, 6), crossing the outer membranetwice (topology 2, 5), or crossing both inner and outer membrane once(topology 3, 4). Protease digestion and antibody epitope mapping werethen used to identify the correct topology (Donzeau et al. 2000)(Hermann et al. 1998). Mitochondria were first purified with a sucrosegradient so that the mitochondria are at least 90% pure (Yuryev et al.2000). By incubating mitochondria in HEPES-mannitol buffer to maintainthe mitochondrial outer membrane intact, a dose-dependent limitedprotease K digestion was performed and analyzed the digestedmitochondria with Ab-N or Ab-1 antibodies. These two antibodiesrecognize the N terminal 50-residue epitope or amino acids 219˜232 inthe region between the two transmembrane domains, respectively. See FIG.4 a. In the Western analysis with Ab-N or Ab-1, the 30 kD PLS3 proteinis digested to a 25 kD fragment. See FIG. 4 b. Similar results wereobtained in a limited protease K digestion from 5 min to 30 min or 4° C.to 25° C. (data not shown). These results suggested that the PLS3protein is accessible to protease K in the intact mitochondria. In otherwords, PLS3 protein has a small portion present at outside ofmitochondria. Topologies 1, 2, 5 could be eliminated based on theseresults.

Purified mitochondria were then incubated with a low concentration HEPESbuffer to generate mitoplasts by osmotic swelling and disruption of theouter membranes. This allows protease K accessible to theintermembranous space of mitochondria, but not to the matrix. Thecleavage products analyzed by Ab-1 Western blot revealed furthercleavage of the 25 kD product to smaller fragments at sizes of 15-25 kD.Western analysis of the undigested outer membrane fraction failed toshow any PLS3 protein (see FIG. 4 c far right lane). These two findingseliminated possibility 6, which is expected to have PLS3 present in theouter membrane fraction. When the protease K cleavage products ofmitoplasts were analyzed with Western blotting by Ab-N, it was observedat the highest concentration of protease K a smallest band at 9 kD whichis still recognized by Ab-N (see FIG. 4 d). This is compatible withtopology 3 with only the first 70 amino acids left after digestion ofthe intermembranous portion of PLS3. Topology 3 was thus confirmed to bethe correct orientation of PLS3 in mitochondria, with the N-terminus atthe matrix side of mitochondria and the C-teminus at outside of theouter membrane of mitochondria.

Topology was also determined by antibody epitope mapping. Intactmitochondria and isolated mitoplasts were used to study the localizationof the two epitopes recognized by two antibodies. Mitochondria ormitoplasts were used for incubation with either of the antibodies. Afterremoving the excessive antibodies by washing, they were incubated with asecondary antibody conjugated with FITC for quantification of firstantibody bound in pelleted mitochondria or mitoplasts. As showed in FIG.4 e, intact mitochondria failed to be recognized by both Ab-1 and Ab-N,suggesting that neither of the epitopes is outside of the mitochondria.On the other hand, mitoplasts were recognized by Ab-1, but not by Ab-N.This result indicates that the epitope between the two TM domains is atthe intermembranous space of mitochondria and the N-terminus is insideof mitoplasts. When mitoplasts were treated with protease K, theantibody recognition by Ab-1 decreased, indicating that the epitoperecognized by Ab-1 is sensitive to protease K and therefore outside ofmitoplasts. All these results are consistent with the topology 3. Bothprotease digestion and epitope mapping studies thus lead to theconclusion that the PLS3 protein crosses both inner and outer membranesof mitochondria with the N-terminus at the matrix of mitochondria. Thisis the second protein reported to cross both mitochondrial inner andouter membranes in addition to Tim23, a subunit of mitochondrialpreprotein translocase complex (Donzeau et al. 2000).

PLS3 protein disrupts mitochondrial transmembrane potential and inducescytochrome c release in vitro.

Using freshly isolated mouse liver mitochondria, an in vitro system wasset up to study PLS3 function. Because PLS3α could not be overexpressedin bacteria, it was investigated whether purified PLS3β proteininterferes with mitochondrial Δψ. Mouse liver mitochondria wereincubated with PLS3β protein and then Δψ was measured with Rhodamine123. The system was tested with calcium as a positive control, sincecalcium is known to induce disruption of Δψ (Duchen 2000). It was foundthat PLS3β protein induced disruption of Δψ in a dose dependent mannersimilar to calcium (FIG. 5 a). The supernatant of PLS3β and mitochondriamixtures was then separated by centrifugation. Cytochrome c release wasnext analyzed with Western blotting. Cytochrome c was present in thesupernatants of PLS3-treated mitochondria but not in buffer-treatedmitochondria (FIG. 5 b). This showed that exogenous PLS3 induceddisruption of Δψ and release of cytochrome c in vitro. These resultsseem to be inconsistent to the in vivo overexpression of PLS3β, whichdid not induce loss of Δψ, and cells were viable with thisoverexpression. Without being limited to any one theory, it is thoughtthat this could be due to a presence of inhibitors in cells, whichprevents PLS3 from disruption of Δψ. The loss of Δψ and lethality ofN-terminal deletion mutant indicate that this inhibitor may mediate itseffect through this N-terminal portion. Another possibility is that PLS3is post-translationally modified, such as phosphorylation in PLS1, tomaintain PLS3 in an inactive state. The existence of two bands in PLS3Western analysis may support this hypothesis. See FIGS. 2 a and 4 b.

PLS3 is a Cardiolipin Transferase In Vitro.

Since PLS3 has sequence homology with human plasma membrane phospholipidscramblase (PLS1) (Wiedmer et al. 2000), a phospholipid transfer assaywas performed to determine the substrate specificity of PLS3. For thispurpose, a purified PLS3β protein was used for an in vitro phospholipidtransfer assay. Whether PLS3 had transferase activities forphosphatidylcholine (PC), phosphatidylethanolamine (PE) and cholesterol(C) using ¹⁴C-labeled phospholipids as substrates was first evaluated.When PLS3β protein was incubated with liposomes containing ¹⁴C-labeledPC and recipient mitochondria, the recipient mitochondria had nodifference in activity compared with BSA-treated mitochondria (FIG. 6a). In contrast, studies with PE and cholesterol showed that PLS3βinhibited the intrinsic phospholipid transferase activities for PE andcholesterol (FIG. 6 a). This finding could be related to the fact thatPLS3 induced disruption of mitochondrial transmembrane potential (FIG. 5a), but the exact mechanism remains to be determined.

Cardiolipin was then investigated since cardiolipin is amitochondrion-specific phospholipid and is potentially more critical inthe process of apoptosis. Because radioactive cardiolipin is notcommercially available, NAO was used to quantify cardiolipin in therecipient mitochondria. NAO binds cardiolipin specifically withoutinterference by other phospholipids with a stoichiometry of 1 to 1 or 2to 1. To achieve maximal NAO saturation, the recipient mitochondria werepermeablized and fixed with formaldehyde. A dose titration of NAO wasthen performed. The red fluorescence achieved a plateau when the NAOconcentrations were above 20 μM as reported (Garcia Fernandez et al.2000). The concentration of 30 μM was thus chosen to determine the totalamount of cardiolipin after in vitro cardiolipin transfer assay. It wasconfirmed that PLS3 increased the total amount of cardiolipin inrecipient mitochondria by 35%, while mitochondria treated with buffer(NC) or bovine serum albumin (BSA) control had similar basal levels ofcardiolipin (FIG. 6 b). These results confirmed that PLS3 functions as amitochondrial cardiolipin transferase.

PLS3 increases the percentage of cardiolipin at the outer membranes ofmitochondria.

Once it was established that PLS3 is a mitochondrial cardiolipintransferase, it was investigated whether overexpression of PLS3 had anyeffect on total mitochondrial cardiolipin in 293 cells. Cells weretransfected with pPLS3-IRES-EGFP or pIRES-EGFP for control and sortedfor GFP positive cells. Western analysis confirmed the overexpression ofPLS3 protein. Total amounts of cardiolipin were determined by NAO usingthe fluorescence intensity at 630 nm to represent cardiolipin amount. Itwas found that GFP positive cells from pPLS3-IRES-EGFP transfection hadno difference in cardiolipin compared with the GFP positive cells fromcontrol pIRES-EGFP transfection (not shown). It was thus suspected thatPLS3 translocates cardiolipin between two intracellular compartments,such as the inner and outer membranes of mitochondria. In this case, thetotal amount of cardiolipin remains unchanged with PLS3 overexpression.

It is known that cardiolipin is mainly at the inner membrane ofmitochondria with the ratio of cardiolipin between the outer and innermembranes about 3:21 or 12% at the outer membranes (Parsons 1967;Parsons and Yano 1967). The amount and redistribution of cardiolipin inthe PLS3 overexpressing cells with or without UV irradiation-inducedapoptosis was then investigated. 293 cells were first tested, and NAOdose titration was performed 10 hours after UV irradiation. In the curveof 530±25 nm, UV irradiation induced an expected decrease in NAO boundcardiolipin (FIG. 7 a left), which is related with peroxidation ofcardiolipin as reported (Shidoji et al. 1999). Fluorescence was thenanalyzed at 680±30 nm, which provides information about cardiolipincomposition at the inner and outer membranes as described (GarciaFernandez et al. 2000). By slowly increasing the NAO concentration,cardiolipin at the outer membrane of mitochondria becomes NAO saturatedfirst, and then NAO enters mitochondria to interact with cardiolipin atthe inner membrane. There is a shoulder after saturation of outermembrane, and the percentage of cardiolipin at the outer membrane couldbe represented by the percentage of cardiolipin at this shoulder versustotal cardiolipin. The percentage of cardiolipin at the outer membraneof mitochondria in 293 cells is about 10% (solid arrowhead), which isbarely visible. With UV irradiation, the shoulder increases to 30% (openarrowhead, FIG. 7 a right). This means that UV irradiation increases theproportion of cardiolipin at the outer membrane of mitochondria.

Cells with PLS3 overexpression were then investigated. At the curve of530±25 nm, there is an early rise of fluorescence up to 10 μM NAO,suggesting that cardiolipin at the outer membrane increases (FIG. 7 bleft). Using 680±30 nm to quantify the percentage, it was observed thatthe shoulder of non-irradiated cells is at 30% (solid arrowhead), whichis higher than the 10% in normal 293 cells. After UV irradiation of PLS3expressing cells, the shoulder further increases to about 50% (openarrowhead, FIG. 6 b right). These results indicated that overexpressionof PLS3 increases the proportion of cardiolipin at the outer membranesof mitochondria and UV irradiation further increases the percentage.

PLS3 Enhances the Apoptotic Effect of Bcl-B.

Bcl-B is a cell death agonist which translocates to mitochondria uponapoptotic stimulation (Lee et al. 2001). In this study, it was foundthat Bcl-B interacts with a mitochondrial enzyme, PLS3. Since thebiochemical functions of most Bcl-2 family members are unclear, findingout whether Bcl-B functions through modulation of PLS3 activity and indetermining the consequence of Bcl-B and PLS3 interaction was importantto evaluate. Equal amounts of plasmids of either or both Bcl-B and PLS3expression vectors were co-transfected into 293T cells. It was observedthat cells transfected with pcDNA vector only or pcDNA-PLS3 plasmids hadno difference in apoptosis compared with non-transfected cells. Cellstransfected with pcDNA-Bcl-B has increased apoptosis as expected (Lee etal. 2001); while cells transfected with both pcDNA-Bcl-B and pcDNA-PLS3αdeveloped significantly more apoptosis than Bcl-B alone (FIG. 8 a).Using propidium iodide, the proportion of sub-G1 cells was quantified,or the apoptotic population, for cells without transfection ortransfected with pcDNA control, pcDNA-PLS3α or pcDNA-Bcl-B as 1.4%,2.8%, 3.8% and 9.8%, respectively. Cells transfected with bothpcDNA-PLS3α and pcDNA-Bcl-B resulted in 15.4% apoptosis. Without beinglimited to any one theory, it appeared that this indicated that the celldeath effect of Bcl-B was enhanced by co-expression of PLS3. In order toeliminate the possibility that difference in the amounts of transfectedDNA contributed to the differences in apoptosis, sequential transfectionwas performed. A clone of GFP positive cells was obtained from apPLS3-IRES-EGFP transfection and transfected this clone withpcDNA-Bcl-B. As a control, pcDNA-Bcl-B were transfected into GFPpositive cells obtained from a pRES-EGFP transfection. Significantlymore apoptotic cells were observed in pcDNA-Bcl-B transfected into PLS3cells (not shown). This result confirmed that expression of PLS3αenhanced the death promoting effect of Bcl-B. Because Bcl-B physicallyinteracts with PLS3, it was thought that Bcl-B may activate PLS3 throughthis interaction. Once PLS3 is activated, cardiolipin translocates inmitochondria and induces disruption of Δψ and cell death.

Activation of PLS3 by Bcl-B.

In order to prove that Bcl-B induces direct activation of PLS3, Bcl-Bexpression vectors were transfected into 293-PLS3 cells and thepercentage of cardiolipin was measured at the outer membrane ofmitochondria. Using the same methods as used in FIG. 7, it was observedthat Bcl-B expression in 293-PLS3 cells shifted the curve of redfluorescence upward as was seen in UV irradiation of 293-PLS3 cells. Thepercentage of the shoulder, which represents the percentage ofcardiolipin at the outer membrane, increase from 30% in 293-PLS3 cellsto 50% in Bcl-B expressers, which is similar to that of UV irradiation(FIG. 7 c). This result supports that Bcl-B induces activation of PLS3.

An enzymatically inactive mutant of PLS3 was constructed to determinewhether this mutant prevents Bcl-B-induced apoptosis. By comparing thesequences of PLS1 and PLS3, a conserved amino acid Phe258 was identifiedwhich is located at a conserved calcium-binding motif of PLS3. Mutationof the corresponding Phe in PLS1 completely abolished the activity ofPLS1 (Zhou et al. 1998). Phe258 was mutated to valine and apCMV-PLS3(F258V) expression vector was constructed. 293 cells were thentransfected with this vector and a stable transfected clone was obtainedwhich has an obvious phenotype of slow growing. Apoptosis was analyzedin pCMV-PLS3(F258V) cells transfected with pBcl-B-IRES-EGFP to comparewith 293-pCMV control cells transfected with the same Bcl-B expressionvector. Cell death was detected in 14% of GFP positive cells in 293-pCMVcontrol cells after transfection. However, only 7.5% of GFP positivecells in PLS3(F258V) expressors were apoptotic. This result indicatesthat PLS3(F258V) inhibits the cell death effect of Bcl-B, and confirmsthat PLS3 is a downstream effector of Bcl-B.

Discussion

Bcl-B-specific functions were then investigated by identifying PLS3 thatinteracts with Bcl-B. In contrast to PLS1, PLS3 is localized inmitochondria and has the substrate specificity to cardiolipin. Tounderstand where PLS3 moves cardiolipin to, the topology of PLS3 inmitochondria was determined. It was found that PLS3 has a rare topologyby crossing both inner and outer membranes of mitochondria. This type ofmitochondrial topology has only been described in Tim23, a proteinoperative in mitochondrial preprotein translocation (Donzeau et al.2000). Tim23 is responsible for translocating pre-protein intomitochondria; while PLS3 is likely responsible for cardiolipintranslocation between inner and outer membranes. These studies appearedto confirm this theory. This study demonstrated a cardiolipin shift fromthe inner membrane to outer membrane by PLS3 during the process ofapoptosis. This is reminiscent of PLS1-related PS translocation in theplasma membrane, and will facilitate more investigations inmitochondrial phospholipid changes during apoptosis.

The consequences of cardiolipin redistribution in mitochondria were alsostudied. Cardiolipin is associated with the major proteins of oxidativephosphorylation, such as ATP synthase, respiratory complex I, II and IV,as well as the carrier protein for phosphate and adenine nucleotides(Schlame et al. 2000). A yeast mutant lacking cardiolipin synthase, thecritical enzyme for making cardiolipin, was viable; but moderatelydeficient in mitochondrial energy transforming machinery at 25° C.(Jiang et al. 2000). When the temperature increased to 37-40° C.,respiration was completely uncoupled from phosphorylation, andmitochondrial transmembrane potential was disrupted (Koshlin andGreenberg 2000). This confirmed the importance of cardiolipin incritical mitochondrial functions.

The fate of cardiolipin during the process of apoptosis has just begunto be addressed. Cardiolipin undergoes peroxidation during apoptosis,and this peroxidation leads to dissociation of cytochrome c fromcardiolipin (Shidoji et al. 1999; Nomura et al. 2000). Cytochrome crelease and apoptosis were inhibited by overexpression of mitochondrialhydroperoxide glutathione peroxidase, which prevented cardiolipinperoxidation (Nomura et al. 2000). Although it is unclear howperoxidation of cardiolipin leads to changes in oxidativephosphorylation and mitochondrial dysfunction, failure of cytochrome cbinding with peroxidized cardiolipin will at least partially contributeto the release of cytochrome c from mitochondria. Otherwise, cytochromec will still be sequestered in the intermembranous space by cardiolipineven the putative cytochrome c channels are wide open.

When cardiolipin is translocated from the inner membrane to the outermembrane of mitochondria, the enzymes and cofactors responsible for theoxidative phosphorylation will be disturbed. This can directly lead to areduction in oxidative phosphorylation. Harris et al. provided evidencesthat Bax induces its death effect by reducing the oxidativephosphorylation and the Bax toxicity is reduced in yeast strainsdeficient in oxidative phosphorylation (Harris et al. 2000). On theother hand, Bcl-xL promotes efficient ADT-ATP exchange to allowmitochondria to adapt to change in metabolic demand to prevent apoptosis(Vander Heiden et al. 1999). These findings support the importance ofoxidative phosphorylation in mitochondria in the process of apoptosis.Redistribution of cardiolipin during apoptosis will thus have a majorimpact in mitochondrial function.

Recently, Lutter et al. identified cardiolipin as the target of tBidduring the process of apoptosis (Lutter et al. 2000). Because verylittle cardiolipin is normally present at the outer membrane ofmitochondria, the translocated cardiolipin by PLS3 provides the requiredtBid receptor at the outer membrane during apoptosis. If PLS3 plays sucha key role in this cardiolipin translocation, inhibition of PLS3activity with a dominant negative mutant will be able to preventapoptosis by a variety of death signals. This appears likely to be thecase. Overexpression of a mutant PLS3(F258V) also preventsapoptosis-induced by UV irradiation in addition to Bcl-B expression(manuscript in preparation).

It was observed that the PLS3 protein disrupted Δψ and inducedcytochrome c release in vitro. However, overexpression of PLS3 did notcause cell death or disrupt Δψ. One possibility is that PLS3 ispost-translationally modified, such as phosphorylation, and renderedinactive in mitochondria. When cells undergo apoptosis, PLS3 becomesactivated. The activated PLS3 subsequently moves cardiolipin inmitochondria and induces mitochondrial apoptotic change. Thepro-apoptotic effect of N-terminal-deletion mutant of PLS3 is similar tothe in vitro results of PLS3 in disrupting Δψ. This suggests that theregulatory effect is mediated through the N-terminal portion of PLS3,perhaps through interaction with an inhibitor in normal mitochondria.Phosphorylation of PLS3 may affect the interaction between PLS3 and theputative inhibitor. According to our topology result, the N-terminus islocated at inner membrane and matrix, which is not accessible by factorsfrom cytosol. The putative inhibitor is thus likely a mitochondrialprotein present in mitoplasts.

From the point of view of a possible role in cell death regulator, PLS3also needs to be in an inactive state in normal cells and becomesactivated upon induction of apoptosis. Otherwise, cells would not beable to keep cardiolipin mainly in the inner membrane, and cells wouldspontaneously lose mitochondrial integrity and undergo apoptosis. Thisis similar to PLS1, which is also inactive in normal cells and becomeactivated by PKC-δ during apoptosis (Frasch et al. 2000). Investigationof the mechanism of PLS3 activation will be a crucial study inregulation of apoptosis. One known mechanism is through interaction ofBcl-B. This idea is supported by our findings that PLS3 and Bcl-Binteract physically, and the combination of Bcl-B and PLS3 greatlyenhanced cell death compared with either PLS3 or Bcl-B alone.Furthermore, both UV-irradiation and Bcl-B expression induce an increasein the percentage of cardiolipin at the outer membrane of mitochondria.This strongly supports that PLS3 can be activated by Bcl-B in triggeringcell death.

According to these observations, a model as shown in FIG. 8 b isproposed. Apoptotic stimulation or Bcl-B translocation induces theactivation of PLS3, which translocates cardiolipin to the outermembranes of mitochondria. This results in at least two consequences.One is to disturb mitochondrial oxidative phosphorylation and poorenergy production, and the other is to help carrying cytochrome c to theoutside of mitochondria for subsequent caspase activation. Thetranslocated cardiolipin also helps to recruit tBid to mitochondria,which is one of the most powerful death factors. The combination of allthese events eventually leads to total mitochondrial breakdown and theeventual cell death.

Experimental Procedures

I. Identification of PLS3 cDNA and Two Alternative Transcripts.

A complete system of human liver cDNA MatchMaker yeast two-hybridlibrary was purchased from Clontech (Palo Alto, Calif.). Bcl-B wascloned in-frame into the pAS2 vector at NcoI and EcoRI sites. ThepAS2-Bcl-B plasmid was transformed into yeast strain PJ69-2A for matingwith the pretransformed library, which was constructed in the Y187 yeaststrain. The mated yeast culture was selected with drop-out media lackingadenosine, histidine, leucine and tryptophan (DO-4 media) as describedin the company protocol. Positive clones were confirmed with B-galcolony-lift assay. All positive clones were grown in liquid culture andplasmids were isolated. They were re-confirmed by growth in DO-4 mediaafter co-transformation with pAS2-Bcl-B into the PJ69-2A strain. Theywere considered false positive if PJ69-2A grew in selection media aftertransformed with plasmids in combination with the pAS2-lamin C controlvector. The final confirmed clones were sequenced and identities wereobtained through Blast search.

Construction of PLS3 Expression Vectors.

PLS3α, PLS3β and ΔPLS3 were inserted in-frame into the SmaI site ofpEGFP-C1 vector (Clontech, Palo Alto, Calif.). PLS3α was also clonedinto Hind m and Bam HI sites of pIRES-EGFP and pcDNA3.1 vector(Invitrogen, Carlsbad, Calif.) for overexpression from a CMV promoter.In order to overexpress PLS3β protein in bacteria, PLS3β was cloned inframe into pGEX6P3 vector (Amersham-Pharmacia, Piscataway, N.J.) at anEcoRI site and expressed the glutathione-S-transferase-PLS3 fusionprotein in BL21 competent cells for affinity purification. PLS3β proteinwas cleaved from GST fusion protein by precision protease according tothe supplier's protocol (Amersham-Pharmacia Biotech. Inc. Piscataway,N.J.).

Northern Analysis and Western Analysis.

The human 12-Lane MTN blots were purchased from Clontech (Palo Alto,Calif.). The PLS3 cDNA was labeled with α³²P-dCTP (NEN) by randompriming and then hybridized with blots according to the companyprotocol. PLS3 antibody was raised in two rabbits against a peptidecorresponding to amino acids 219-232 of PLS3 with the sequenceCDTNFEVKTRDESRS (SEQ ID NO: 8). The peptides were conjugated to beads byEDC/Diaminodipropylamine Immobilization kit (Pierce, Rockford, Ill.) toprepare an affinity column. Antibody was purified by the affinity columnaccording to the company protocol. Western analysis was performed bystandard procedure with PLS3 antibody at 1:100 dilution ofaffinity-purified primary antibody and 1:1000 of goat anti-rabbitsecondary antibody conjugated with horseradish peroxidase(Amersham-Pharmacia Biotech. Inc. Piscataway, N.J.). The blots weredeveloped with chemiluminescence for autoradiography (Pierce, Rockford,Ill.).

Mitochondria and Mitoplasts Preparation.

Mitochondria were isolated by differential centrifugation. In brief,cells were treated with a buffer containing 300 mM sucrose, 10 mM Tris(pH 7.5), 5 mM EDTA, and 1 mM PMSF protease inhibitor for 5 min on ice.Cells were broken by passage through 25 G needle for 10 strokes. Nucleiand unbroken cells were collected by centrifugation at 1000×g for 5minutes. Mitochondria were then collected by centrifugation at 10,000×gfor 10 minutes. Microsomal fractions were pelleted byultracentrifugation at 30,000×g for 1 hour. The supernatants of thefinal centrifugation were saved for cytosolic fractions. For furtherpurification of mitochondria for topology study, crude mitochondrialfraction was separated by a sucrose gradient at 1-2 M in mitochondrialisolation buffer. Mitoplasts were prepared by osmotic disruption ofouter membrane with 10 mM HEPES/KOH, pH 7.4. After incubation on ice for20 min and stirring to remove outer membranes, mitoplasts were collectedby 20,000×g for 15 min (Hermann et al. 1998).

In vitro measurement of mitochondrial transmembrane potential andcytochrome c release.

Purified mouse liver mitochondria were quantified by the amount of totalprotein. Before analysis of transmembrane potential, mitochondria werefurther washed with the isolation buffer without EDTA. For each in vitroassay, 100 μg of mitochondria were incubated with 200 nM Rhodamine 123(Molecular Probe, Eugene, Oreg.) and fluorescent intensities at 585 nmwere determined by FL600 fluorescence microplate reader (Bio-TekInstrument, Inc.). Mitochondria were treated with 100 nM calcium aspositive control or different amount of purified PLS3 protein for 10 minbefore the assay. The mixture of mitochondria and PLS3 protein wascentrifuged at 10,000×g for 10 min to separate supernatants andmitochondrial pellets. The supernatants were subsequently analyzed byWestern for cytochrome c.

Phospholipid Transfer Assay.

Phospholipid transfer assay was modified from Koumanov et al. (Koumanovand Infante 1986). In brief, phospholipids were dissolved in chloroform,mixed and evaporated to dryness with a stream of nitrogen. Driedphospholipids were resuspended with the same buffer used for isolatingmitochondria and sonicated in water bath for 5 min. Liposomes, whichcontained 280 nmole cold phospholipids and 1 μl ¹⁴C-labeledphospholipids (NEN) for each reaction, were mixed with freshly-preparedmouse liver mitochondria along with purified PLS3β or bovine serumalbumin (BSA) as control, for 20 minutes at 37° C. Mitochondria werethen washed five times with mitochondrial preparation buffer to removefree liposomes and counted by scintillation counter for ¹⁴C activity.For cardiolipin transfer assays, total amounts of cardiolipin werequantified by specific fluorescence with NAO dye. NAO (30 μM) was addedto recipient mitochondria, and fluorescence intensity was measured at590±35 nm by fluorescence microplate reader.

Flow Cytometry Analysis.

Nonyl acridine orange (NAO) dye, [10-N-nonyl-3,6-bis (dimethylamino)acrydine], was purchased from Molecular Probe Inc. (Eugene, Oreg.).Cells were fixed with 1% formaldehyde for 15 minutes at room temperatureand washed twice with cold phosphate buffered saline (PBS) beforestaining with 30 μM NAO. Cells were fixed with 95% of alcohol at 4° C.overnight before staining with 50 μM of propidium iodide for cell cycleanalysis. AnnexinV-PE was used for apoptosis assay according to companyprotocol (PharMingen). Flow cytometry analyses were performed withFACScan cytometer (Becton Dickinson, San Jose, Calif.).

II. Phospholipid Scramblase 3 is a Member of the Scramblase Family thatis Present in the Mitochondria

Identification of PLS3α

A MatchMaker yeast two-hybrid kit was purchased from Clontech (PaloAlto, Calif.). The cDNA of hBoo was inserted in-frame into the pAS2vector and transformed into yeast strain PJ69-2A for mating with theY187 pretransformed human liver cDNA library. The mated diploid yeastwas selected with media lacking adenosine, histidine, leucine andtryptophan (DO-4 media). Positive clones were confirmed withβ-galactosidase assay. Clones were re-confined by growing in DO-4 mediaafter co-transformation with pAS2-hBoo into the PJ69-2A strain. Theplasmid pAS2-lamin C was used to rule out non-specific interaction. Thefinal confirmed clones were sequenced and identities were obtainedthrough the BLAST search. The cDNA of PLS3 was inserted into pEGFP-C1vector (Clontech) and pcDNA3.1 vectors (Invitrogen) for mammalianexpression.

Western Analysis

PLS3 antibodies, Ab-1 and Ab-N, were raised in rabbits with a peptidecorresponding to amino acids 219-232 of PLS3 (CDTNFEVKTRDESRS, SEQ IDNO: 8) and recombinant PLS3(amino acids 1-50), respectively. Antibodieswere purified by the corresponding affinity columns. Western blotanalysis was performed by standard procedures with PLS3 antibodies at1:250 dilution and developed with chemiluminescence.

Subcellular fractionation and preparation of mitochondria andmitoplasts.

Mitochondria were isolated by differential centrifugation. In brief,mouse liver was incubated in mitochondrial isolation buffer containing300 mM sucrose, 10 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% BSA and 1 mMPMSF for 5 min on ice. Cells were disrupted by douncing 10 strokes, andcentrifuged at 1000×g for 5 min, 10,000×g for 10 min, and 30,000×g for60 min, for collection of intact cells/nuclei, crude mitochondria andmicrosomes, respectively. The final supernatants were used ascytoplasms. To further purify mitochondria for topology study, crudemitochondria were loaded on a sucrose gradient of 1-2 M in themitochondrial isolation buffer, and centrifuged at 100,000×g for 90 minin a table-top ultracentrifuge (Beckman OptimaTMMax). Mitoplasts wereprepared by osmotic disruption of the mitochondrial outer membrane (OM)with 10 mM HEPES/KOH (pH 7.4), and centrifuged at 20,000×g for 15 min.To determine the purity of separation, we measured the activities ofmonoamine oxidase (MAO) and malate dehydrogenase (MDH), as OM and innermembrane (IM) markers (Ragan, 1995). The proteinase K digestion wasperformed as described (Donzeau et al., 2000).

Epitope Mapping of PLS3

Mitochondria or mitoplasts (200 μg) were incubated with Ab-1 or Ab-N at1:100 for 30 min, and then with secondary antibody conjugated withfluorescein isothiocyanate (FITC) at 1:100 for 30 min. The fluorescentintensity of the pellet-bound FITC was measured by FL600 fluorescencemicroplate reader.

III. Targeting Phospholipid Scramblase 3 Disrupts MitochondrialStructure and Function

Results

Both wild-type and mutant PLS3 localize to mitochondria

To investigate its function in mitochondria, we targeted PLS3 bymutating its calcium-binding motif that is highly conserved among PLSfamily members (Zhou et al., 1998). This approach was successfullyemployed by Zhou et al (Zhou et al., 1998), who found that mutation ofPhe281 in PLS1 abolished function. By site-directed mutagenesis, weconverted the corresponding Phe258 in PLS3 to valine, and generatedstable transfectants of the mutant PLS3(F258V) in HEK293 cells.Transfectants of control (293-vector) and wild-type PLS3 (293-PLS3) weresimilarly prepared. Whole cell lysates of the G418-resistant clones wereexamined by Western blotting. Endogenous PLS3 could be detected incontrol whole cell lysates, and transfectants expressing the wild-typeor mutant PLS3 demonstrated increased PLS3 levels (FIG. 13 a).Subcellular fractionation indicated that the PLS3(F258V) proteinlocalizes to the mitochondria, similar to the wild-type PLS3 (FIG. 13b). The integrity of cytosolic and mitochondrial fractions was confirmedby blotting for tubulin and voltage dependent anion channel (VDAC),respectively (FIG. 13 b). Similar transfectants were established in HeLacells (not shown) for additional experiments described below.

Slow Growth of Cells Expressing PLS3(F258V) Mutant

293-vector, 293-PLS3, and 293-PLS3(F258V) cells were cultured over a3-day period and serial cell counts were performed to monitor cellproliferation. The growth rate of 293-PLS3 cells was comparable to thatof 293-vector cells, but 293-PLS3(F258V) cells grew at a slower rate(FIG. 13 c), indicating that the PLS3(F258V) mutant interferes with cellgrowth. Cell cycle analysis revealed that the slow growth did not resultfrom spontaneous G1 or G2/M arrest (not shown). Next, we incubated thesecells in the presence of Na₃N, an uncoupler of oxidative phosphorylation(Simbeni et al., 1990), and compared their growth. The same slow growthrate was observed at three different Na₃N concentrations, as the slopeof all the growth curves was similar to that of the 293-PLS3(F258V)cells without Na₃N (FIG. 13 d).

Targeting PLS3 Decreases Mitochondrial Mass, Transmembrane Potential,and Oxidative State

Because PLS3 localized to mitochondria and the growth of 293-PLS3(F258V)cells was not affected by uncoupling of oxidative phosphorylation, wesuspected that the mitochondria might be defective in PLS3-targetedcells. First, we analyzed the mitochondrial mass and membrane potentialusing JC-1 dye and flow cytometry. There were two peaks in JC-1 greenfluorescence, corresponding to the mitochondrial mass (Camilleri-Broetet al., 1998). The low intensity peak was predominant in 293-vectorcells and 293-PLS3(F258V) cells, while the higher intensity peak wasmore prominent in 293-PLS3 cells (FIG. 14 a, left). Analysis of JC-1 redfluorescence, corresponding to the mitochondrial transmembrane potential(Camilleri-Broet et al., 1998), revealed a relative right shift in293-PLS3 cells and left-shift in 293-PLS3(F258V) cells (FIG. 14 a,right). Thus, while over-expression of PLS3 was associated withincreased mitochondrial mass and transmembrane potential, expression ofPLS3(F258V) was associated with decreased mitochondrial mass andtransmembrane potential.

Next, we measured the oxidative state of these cells using reducedRosamine and flow cytometry. Over-expression of PLS3 was associated witha right-shift, indicating an increased oxidative state, compared tocontrol cells (FIG. 14 b). By contrast, expression of PLS3(F258V) wasassociated with a left-shift, or decreased oxidative state (FIG. 14 b).The relatively decreased oxidative state of 293-PLS3(F258V) cells isconsistent with the lack of inhibitory effect of Na₃N on growthdescribed above.

Finally, we quantitated in these cells several mitochondrial markersthat relate to oxidative phosphorylation. As shown in FIG. 14 c, levelsof cytochrome c were dramatically reduced in 293-PLS3(F258V) cellscompared to 293-vector and 293-PLS3 cells. By contrast, levels of VDACwere unaffected in 293-PLS3(F258V) cells and slightly increased in293-PLS3 cells (FIG. 14 c). Staining for cytosolic tubulin served as aloading control (FIG. 14 c). Given the interaction of cytochrome c withmitochondrion-specific cardiolipin(Shidoji et al., 1999), we determinedthe relative amounts of cardiolipin using the cardiolipin-specificfluorescence dye NAO. As shown in FIG. 14 d, cardiolipin levels werereduced in 293-PLS3(F258V) cells by almost 50% compared to control orPLS3-over-expressing cells.

Targeting PLS3 reduces intracellular ATP and mitochondrial respiration.

The slower growth and reduced oxidative state of 293-PLS3(F258V) cellssuggests that PLS3 targeting interferes with mitochondrial respiration.The total intracellular ATP levels were measured by preparingtrichloroacetic acid (TCA)-treated cell lysates for a luciferase assay(Lundin, 2000). While the ATP concentration was 15% higher in 293-PLS3cells compared to 293-vector cells, it was 10% lower (p<0.01) in293-PLS3(F258V) cells (FIG. 15 a).

Next, oxygen consumption was measured in isolated mitochondria uponincubation with succinate (substrate for state 4 respiration) andsubsequently after addition of ADP (for state 3 respiration). Comparedto control and 293-PLS3 cells, the rate of oxygen consumption wasreduced in 293-PLS3(F258V) cells (FIG. 15 b). The rate of state 4respiration was slightly lower in 293-PLS3(F258V) cells (3.5±0.14pmol/min/μg) compared to 293-vector (4.05±0.21 pmol/min/μg) and 293-PLS3cells (4.15±0.21 pmol/min/μg) (FIG. 15 c). The rate of state 3respiration in HEK293-PLS3 cells (17.5±0.71 pmol/min/μg) was higher thancontrol cells (15±1.41 pmol/min/μg), while that of 293-PLS3(F258V) cellsdecreased by nearly 40% (9.85±0.21 pmol/min/μg) (FIG. 15 c). Thusmitochondrial respiration was dramatically suppressed by expression ofthe PLS3(F258V) mutant and slightly enhanced by over-expression ofwild-type PLS3.

Gross Alterations in Mitochondrial Morphology in 293-PLS3(F258V) Cells

The mitochondria were next examined in these cells by electronmicroscopy. Mitochondria in 293-PLS3 cells were distinct from those in293-vector cells, with fewer cristae present (FIG. 16 a,b). By contrast,293-PLS3(F258V) cells displayed few mitochondria and these were notablylarge in size and abnormal in shape (FIG. 16 c). They contained numerouscristae tightly packed together (FIG. 16 c). Thus perturbation of PLS3,either by over-expression or expression of the F258V mutant, causesabnormal mitochondrial morphology.

PLS3 Targeting Suppresses UV-Induced Apoptosis

Given the central role of mitochondria in apoptosis (Brenner andKroemer, 2000), it was determined how targeting PLS3 might affectsusceptibility to apoptosis. HeLa cell transfectants Hela-vector,Hela-PLS3 and Hela-PLS3(F258V) were UV-irradiated and then assessedafter 4 hours for viability using MTT assay. Cell viability after UVirradiation was 50% for HeLa-vector cells, 19.5% for HeLa-PLS3 cells,and 74% in HeLa-PLS3(F258V) cells (FIG. 17 a). To confirm that the celldeath was indeed apoptotic in nature, cells were also examined byAnnexin V staining. As shown in FIG. 17 b, UV irradiation increased thepercentage of Annexin V-positive cells from 16% to 31% in HeLa-vectorcells and from 20% to 38% in HeLa-PLS3 cells. By contrast, minimalchange (11% to 15%) was detected in HeLa-PLS3(F258V) cells after UVirradiation (FIG. 17 b). Thus over-expression of PLS3 enhancedUV-induced apoptosis, while targeting PLS3 was associated withresistance to apoptosis.

Next, the effects of this UV treatment on mitochondrial mass andpotential were determined. As shown above (FIG. 14 a), unirradiated293-vector cells exhibit two populations based on JC-1 greenfluorescence that reflect differences in mitochondrial mass. Exposure toUV did not dramatically change the distribution of these two population(FIG. 17 c). However, the JC-1 red analysis of the mitochondrialpotential exhibits a shift of the curve to left after UV irradiation. In293-PLS3 cells, on the other hand, the JC-1 red curve shifts to rightafter UV (FIG. 17 c). In 293-PLS3(F258V) cells, this pattern did notchange after UV treatment (FIG. 17 c), consistent with the resistance ofthese cells to UV-induced apoptosis.

UV irradiation and PLS3 over-expression increase cardiolipin inmitochondrial OM.

To investigate a potential mechanistic role of PLS3 in apoptosis,mitochondrial phospholipid changes were examined in cells exposed to UVradiation. Cellular phospholipids were labeled with 32P-orthophosphate,and phospholipids were analyzed by thin layer chromatography frommitochondrial membranes. The efficacy of separation of mitochondrial IMand OM was assessed by the marker enzymes MAO and MDH (Daum et al.,1982; Ragan, 1995). Both IM and OM fractions contained about 80% of therespective marker enzymes (FIG. 18 a), similar to what has been reportedin the literature (Ragan, 1995). The most abundant phospholipids,phosphatidylcholine (PC) and phosphatidylethanolamine (PE), did notchange after UV radiation (FIGS. 18 b,c) and the percentages ofcardiolipin were normalized to the levels of PE. In 293-vector cells,12.2% of the total cardiolipin (CL) was present in the OM, and UVtreatment increased this percentage to 27.8% (FIGS. 18 b,d). In 293-PLS3cells, the percentage of CL in the OM was elevated at 22% and furtherincreased following UV radiation to 45% (FIGS. 18 c,d), suggesting thatPLS3 facilitates CL transport from IM to OM at steady state and duringapoptosis. By contrast in 293-PLS3(F258V) cells, there was minimalcardiolipin in the OM that did not increase after UV treatment (FIGS. 18c,d), suggesting that targeting PLS3 prevents mitochondrial CLtransport.

PLS3 Regulates tBid-Induced Mitochondrial Cytochrome c Release

Given that CL is the mitochondrial target of tBid (Lutter et al., 2000),it was hypothesized that PLS3-mediated CL transport to the mitochondrialOM may regulate susceptibility to t-Bid-induced apoptosis. Mitochondriaisolated from 293-vector, 293-PLS3, and 293-PLS3(F258V) cells wereincubated with recombinant t-Bid, and the released cytochrome c wereanalyzed by Western blotting. Mitochondria from 293-PLS3 cells releasemore cytochrome c, or more sensitive to tBid-induced apoptosis thancontrol mitochondria. By contrast, mitochondria from 293-PLS3(F258V)cells release less cytochrome c, or less sensitive to tBid than control.The loading controls by the remaining cytochrome c in the mitochondrialpellets are roughly equal (FIG. 19 a). Using densitometry, thepercentages of cytochrome c release were calculated to be 32.9% in293-vector, 34.6% in 293-PLS3 and 18.9% in 293-PLS3(F258V), respectively(FIG. 19 b). The amount of SMAC that is released after tBid incubationwas also checked (FIG. 19 c). There is also more SMAC released from293-PLS3 mitochondria and less SMAC released from 293-PLS3(F258V) cells,confirming that changing CL in mitochondrial OM affects the sensitivityto tBid-induced apoptosis.

Discussion

The functional characterization of PLS3 in mitochondria usingoverexpression of wild-type or mutant PLS3 is disclosed herein. Themutant PLS3 was constructed by mutation of a conserved calcium-bindingmotif similarly to an inactive mutant established in PLS1 (Zhou et al.,1998). Mutant PLS3 is localized in mitochondria similar to wild-typePLS3. Cells expressing mutant PLS3 have profound morphologic andfunctional changes in their mitochondria. Morphologically, theirmitochondria are fewer but larger, and have densely packed cristae,which is in contrast to the loose cristae in mitochondria expressingwild-type PLS3. Functionally, mitochondria expressing the PLS3(F258V)mutant have defective oxidative respiration and poor oxygen consumption.These findings are very helpful to understand the function of PLS3 inmitochondria.

This kind of abnormal mitochondria in 293-PLS3(F258V) cells has not beenreported to our knowledge. The tightly packed cristae (FIG. 16 c)suggest that the OM may be defective in expansion along with anincreased IM expansion, thereby forcing the IM packed into such apattern. This is in contrast to cells expressing wild-type PLS3, whichdisplayed very loose cristae in their mitochondria compared to control(FIG. 16 b), exactly the opposite of mitochondria with mutant PLS3.Based on morphologic features and the evidence that PLS3 transfers CL(and possibly other phospholipids untested in this study) from the IN toOM, it is suspected that mitochondrial membrane expansion is initiatedfrom the IM and transported to the OM by PLS3. This theory is supportedby IM localization of CL synthase (Schlame et al., 2000). When thephospholipid translocation between the IM and OM is interfered byPLS3(F258V), the mitochondrial OM can not expand properly, which resultsin the observed morphology. Therefore, the function of PLS3 is essentialto mitochondria.

The functional defect of mitochondria expressing mutant PLS3 isapparently related with their lower amounts of CL and cytochrome c. Theamount of both cytochrome c and CL in cells expressing PLS3(F258V)mutant decreased by nearly 50%, but VDAC, which is unrelated tooxidative phosphorylation, did not change. The consequence of low CL torespiration has been shown in a yeast mutant lacking CL. Moderatedeficiency in mitochondrial oxidative phosphorylation was noted in yeastwith no CL synthase growing at 25° C. When the temperature was shiftedto 40° C., respiration was completely uncoupled from oxidativephosphorylation (Koshkin and Greenberg, 2000). The loss of cytochrome cin cells expressing PLS3(F258V) mutant further enhances the defects inoxidative phosphorylation.

The two populations of cells in the flow cytometry study with JC-1 dyesuggest that the activity of PLS3 is related with mitochondrial mass andpotential. Normal cells may have variations in PLS3 activities. HigherPLS3 activity shifts the mitochondrial mass to a higher level (FIG. 14a), and increases mitochondrial potential after UV irradiation, which isdifferent from losing the potential in control 293-vector cells (FIG. 17c). Suppression of activity of PLS3 shifts the mitochondrial mass to alower level (FIG. 14 a). The population with the larger mitochondrialmass appears to be more metabolic active in oxidative phosphorylationand ATP production, as suggested by higher ATP and state 3 respirationin HEK293-PLS3 cells. The population with the lower mitochondrial massappeared less active with lower ATP and oxygen consumption. Therefore,there is a direct correlation between the activity of PLS3 andmitochondrial respiration, and between the activity of PLS3 andsensitivities to apoptosis.

Since the levels of PLS3 affect the sensitivity to apoptosis, it is veryinteresting to understand how PLS3 and CL distribution affect apoptosis.Using NAO staining of CL, Garcia and Fernandez showed that cardiolipinredistribution is an early event of apoptosis (Garcia Fernandez et al.,2002). 32P-labeling of phospholipids and biochemical fractionation ofmitochondrial IM and OM were used to confirm that PLS3 translocates CLfrom mitochondrial IN to OM (FIG. 18), which is enhanced duringUV-induced apoptosis and PLS3 overexpression, and suppressed byexpression of mutant PLS3.

Cardiolipin translocation to mitochondrial OM is significant for severalreasons. First, cardiolipin is the mitochondrial target for tBidrecruitment during apoptosis (Lutter et al., 2000). Second, cardiolipinactivates the membrane permeabilization effect of Bax (Kuwana et al.,2002). Due to the localization of CL synthase (Hoch, 1992; Schlame etal., 2000), CL is mainly (estimated 90%) present in the mitochondrialIM. Thus the only two ways for tBid or Bax to get in contact with CL arethat both proteins penetrate the OM, or that they interact with CL thatis translocated of to the OM. Our result that UV irradiation and PLS3overexpression increase CL in the OM supports the latter. The increasedamount of CL in the OM facilitates tBid recruitment and Bax activation,which was confirmed by the enhanced tBid-induced apoptosis in 293-PLS3cells (FIG. 19 a). The degree of enhancement, however, is small due tothe fact that tBid is already very powerful in inducing apoptosis.Mitochondria from 293-PLS3(F258V) cells release less cytochrome c andSMAC than those from 293-vector control in the same condition,supporting that they are more resistant to apoptosis as predicted.Hence, the regulation of PLS3-dependent CL translocation between themitochondrial IM and OM plays a role in tBid-related apoptosis.

Materials and Methods

Wild-Type PLS3 and PLS3(F258V) Transfectants

HEK293 cells and HeLa cells were maintained in DMEM supplemented with10% fetal calf serum and penicillin/streptomycin. Mutation of Phe258 inPLS3 to valine was carried out by site-directed mutagenesis according tomanufacturer's protocol (Clontech, Palo Alto, Calif.). The cDNAsencoding wild-type PLS3 and PLS3(F258V) were cloned into the expressionvector pcDNA (Invitrogen, Carlsbad, Calif.), and transfected into HEK293or HeLa cells using the calcium phosphate precipitation method.Transfected cells were selected with G418 (1 mg/ml) and resistant cloneswere picked and expanded for Western analysis using anti-PLS3 antibody.Polyclonal antibody was raised in rabbits against the N-terminal 50amino acids of PLS3 (Zymed laboratory, South San Francisco, Calif.) andpurified by affinity chromatography before use.

Flow Cytometry Analysis

Flow cytometry was performed on a FACScan using Cell Quest software(Becton Dickinson, San Jose, Calif.) at the University of Utah corefacility. To determine mitochondrial mass and transmembrane potential,cells were incubated with 10 μg/ml JC-1 or 200 nM Rosamine (MolecularProbes, Eugene, Oreg.) followed by flow cytometry according to themanufacturer's instructions. Cell cycle analysis was performed onethanol-fixed cells stained with propidium iodide (50 μg/ml) followed byflow cytometry analysis.

Subcellular Fractionation and Preparation of Mitochondria and Mitoplasts

Mitochondria were isolated by differential centrifugation. Briefly,mouse liver was incubated in buffer containing 300 mM sucrose, 10 mMTris-HCl (pH 7.5), 5 mM EDTA, 0.1% BSA and 1 mM PMSF for 5 min on ice.Cells were disrupted in a homogenizer by douncing 10 strokes, andcentrifuged 1000×g for 5 min, 10,000×g for 10 min, and 30,000×g for 60min, for collection of intact cells/nuclei, crude mitochondria andmicrosomes, respectively. The final supernatant represented thecytoplasmic fraction.

To further purify mitochondria for outer and inner membrane isolation,crude mitochondria were loaded on a sucrose gradient of 1-2 M in themitochondrial isolation buffer, and centrifuged at 100,000×g for 90 minin a table-top ultracentrifuge (Beckman OptimaTMMax). Mitoplasts wereprepared by osmotic disruption of the mitochondrial outer membrane (OM)with 10 mM HEPES/KOH (pH 7.4), and centrifuged at 20,000×g for 15 min.To determine the purity of separation, the activities of monoamineoxidase (MAO) and malate dehydrogenase (MDH) were measured, as OM andinner membrane (IM) markers (Ragan, 1995).

Cardiolipin and Phospholipid Analysis

For cardiolipin quantitation, cells were fixed with 4% formaldehyde inPBS for 10 min. Cardiolipin was stained with 30 uM [10-N-nonyl-3,6-bis(dimethylamino) acridine orange] (NAO, Molecular Probes) and thefluorescence intensities at 590 nm were measured using a Bio-Tekmicroplate reader. In pilot studies, it was found that an NAOconcentration of 15 μM was sufficient to saturate mitochondrialcardiolipin in 105 cells. For phospholipid analysis, cells were fixedwith 1% formaldehyde for 15 min at room temperature and washed twicewith cold PBS. Phospholipids were extracted according to the standardmethod of Bligh and Dyer (Bligh, 1959). Lipids were analyzed by TLC in asolvent system of chloroform-methanol-water-ammonium hydroxide120:75:6:2 (v/v) (Fine and Sprecher, 1982) using Whatman silica TLCplates (Fisher Scientific, Pittsburgh, Pa.).

Cell Viability and Apoptosis

Cells were irradiated with unfiltered UV-B lamps (National BiologicalCorp., Twinsburg, Ohio) at 4 J/m²/sec over 2 min. Cell viability assaysusing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT, Chemicon International Corp. Temecula, Calif.) were performed asdescribed (Mosmann, 1983). Staining with Annexin V-PE (BD Pharmingen,San Diego, Calif.) and flow cytometric analysis was performed accordingto the manufacturer's recommendations.

Intracellular ATP and Oxygen Consumption

For ATP quantitation, cells (1×107) were washed with cold PBS, lysedwith 5% TCA, and the resulting supernatants were analyzed using acommercial ATP kit (Biotherma, Sweden) and a MLX Microtiter plateLuminometer (Dynex Technologies, Inc). State 3 and 4 mitochondrialoxygen consumption was measured at 250 C using a Mitocell connected to atwo-channel dissolved oxygen measuring system (model #782, StrathkelvinInstrument, Glasgow, UK). Mitochondria were isolated from cells asdescribed (Gottlieb et al., 2002). Briefly, cells were washed once withmitochondria isolation buffer (MIB) containing 200 mM mannitol, 70 mMsucrose, 1 mM EGTA, and 10 mM Hepes (pH 7.4). After incubating 10 min inice-cold MIB containing 0.5 mg/ml BSA, cells were then homogenized usinga syringe-driven 25 G needle. The homogenate was spun at 800×g for 10min at 40 C. The supernatant was collected and spun at 10,000×g for 10min at 40 C, and the pellet was resuspended in MIB containing BSA.Mitochondrial fractions (50 μg protein) were then diluted in respirationbuffer (225 mM manitol, 70 mM sucrose, 10 mM KH₂PO₄ and 1 mM EGTA, pH7.2). For state 4 (steady state) respiration, succinate (Sigma) wasinjected into the chamber at a final concentration of 7 mM and oxygenconcentrations were monitored for 2 min. To measure state 3(ADP-stimulated) respiration, ADP (Sigma) was then added at a finalconcentration of 150 μM for another 6 min.

Electron Microscopy

Cells were fixed overnight at 4° C. in 0.1 M sodium cacodylate with 2.5%glutaraldehyde and 1% paraformaldehyde, with the addition of 2.4%sucrose and 8 mM calcium chloride, then washed twice with 0.1 M sodiumcacodylate and re-fixed for 45 min in 2% osmium tetroxide in 0.1 Mcacodylate buffer. After washing in water, cells were stained enblocwith saturated aqueous uranyl acetate for 1 hour. After dehydration in agraded series of ethanol, infiltration was carried out through a seriesof ethanol: Spurr's plastic ratios and finally embedded in straightSpurr's plastic. Blocks were polymerized overnight in a 60° C. oven, andsectioned with a diamond knife to a thickness of 60-80 nm, and sectionspicked up on 135 hex mesh grids. Sections were stained in saturatedaqueous uranyl acetate followed by Reynold's lead citrate. All electronmicrographs were taken on a Hitachi H-7100 transmission electronmicroscope at 75 KV.

IV. Phospholipid Scramblase 3 is the Mitochondrial Target of PCK-δInduced Apoptosis

Materials and Methods:

HEK293 and HeLa cells were grown in DMEM supplemented with 10% fetalbovine serum. The cDNAs of PLS3 and PLS3(F258V) were cloned intopcDNA3.1 vector (Invitrogen, Carlsbad, Calif.) for expression inmammalian cells. PKC-δ was cloned into pCMV/Mito/myc vector (Invitrogen,Carlsbad, Calif.) to construct the mitochondrial targeted PKC-δ. Go6976,recombinant PKC-δ enzyme and c-abl antibody were purchased fromCalbiochem (San Diego, Calif.). Antibodies against phosphoserine (PS),phosphothreonine PT), phosphotyrosine (PY), PKC-δ and the PKC-δinhibitor, rottlerin, were purchased from Sigma (St. Louis, Mo.).

In Vitro Phosphorylation and Kinetics Assay.

PLS3 protein was overexpressed as His-tagged protein by pQE (Qiagen)vector, and then purified with nickel-column in 8M urea. The PLS3protein was eluted in urea and dialyzed against 2M urea before usage.Immediately before in vitro phosphorylation, PLS3 was further diluted toless than 0.2 M urea in the final phosphorylation reaction. In vitrophosphorylation was performed with 0.1 μg PKC-δ, γ-[32P]-ATP and 1 μg ofrecombinant PLS3 protein in a reaction buffer as described in themanufacturer's protocol (Calbiochem, San Diego, Calif.). The reactionmix was incubated in room temperature and stopped at various timepoints, or after 20 min in the kinetics study, with SDS loading buffer.The phosphorylated products were separated by SDS-PAGE and analyzed byautoradiography.

Immunoprecipitation.

Immunoprecipitation was performed with affinity purified PLS3 antibodyat 1:100 dilution followed by Western analysis with PS, PT, PY and PKC-δantibodies. The Western blot was developed with chemiluminescence(Pierce, Rockford, Ill.).

Apoptotic Assays.

Apoptotic assays were performed with annexinV-PE (BD-PharMingen) permanufacturer's protocol. The TUNEL assay (terminaldeoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) wasperformed according to the manufacturer's protocol (Roche, Switzerland).

Subcellular Fractionation.

Subcellular fractionation was performed by differential centrifugation.In brief, 107 cells were incubated in buffer containing 300 mM sucrose,10 mM Tris (pH 7.5), 5 mM EDTA, 0.1% BSA and 1 mM PMSF proteaseinhibitor for 5 min on ice. Cells were disrupted by passage through a 25G needle or dounced for 10 strokes, and then were centrifuged at 1000×gfor 5 minutes, 10,000×g for 10 minutes, and 30,000×g for 60 minutes, forcollection of intact cells/nuclei, crude mitochondria and microsomes,respectively. The final supernatants were used as cytosols. Themitochondria and the cytosolic fractions were analyzed for cytochrome crelease with Western blot.

Immunofluorescent Staining.

Cells were fixed with 4% paraformaldehyde in phosphate-buffered saline(PBS) for 5 min. Cells were washed and permeabilized with 0.2% TritonX-100 in PBS for 2 min. Non-specific reaction was blocked with 3% BSAfor 15 min. Primary antibody against PKC-δ was used at 1:100 andsecondary antibody with FITC was used at 1:200 dilution. The stainedcells were visualized by Nikon fluorescence microscope Eclipse TE300.

Results

PLS3 is a phosphoprotein and the phosphorylation increases after UVirradiation.

In order to investigate the regulation of PLS3 activity, thephosphorylation status of PLS3 during UV-induced apoptosis were studied.Immunoprecipitation (IP) in HEK293 and HEK293-PLS3 cellular extractswere performed with an antibody to PLS3, and probed the Western blotwith antibodies specific for phosphoserine (PS), phosphothreonine (PT)and phosphotyrosine (PY). The immunoprecipitated PLS3 was recognized bythe anti-PT antibody, but not by anti-PS or anti-PY (FIG. 20 a),indicating that PLS3 is phosphorylated at the threonine residue. UVirradiation increased the PT signals in both the control HEK293 andHEK293-PLS3 cells. The same blot was probed with PLS3 antibody as aloading control, and revealed minimal difference in the amount of PLS3protein before and after UV irradiation (FIG. 20 a).

PKC-δ Physically Interacts with PLS3.

The kinase that phosphorylates PLS3 was then sought. Based on theobservation that PLS1 is phosphorylated by PKC-8, we studied whetherPLS3 is the mitochondrial substrate of PKC-δ. It was first reconfirmedthat PKC-δ is essential for UV-induced apoptosis by blocking PKC-δ witha PKC-δ inhibitor, rottlerin, in our system. The HEK293 cells werefractionated and the isolated mitochondria and cytosols were analyzedfor cytochrome c release by Western blot. This study confirmed thatcytochrome c was released to cytosol after UV irradiation, and thisrelease was inhibited by rottlerin (FIG. 20 b). The loading controls ofthe mitochondria or cytoplasms showed equal amounts of voltage-dependentanion channel (VDAC) or tubulin. To examine the effect of translocatedPKC-δ to mitochondrial cytochrome c release, mitochondria were washedwith mitochondrial isolation buffer to remove rottlerin. The washedmitochondria were then treated with phorbol ester PMA to achieve maximalactivation of PKC-δ, and the released cytochrome c after PMA treatmentwas studied. The mitochondria from non UV-irradiated cells had veryminimal cytochrome c release, indicating very little PKC-δ present. Themitochondria from UV-irradiated but not rottlerin-treated cells releasedthe most cytochrome c after PMA treatment, confirming the existence ofPKC-δ in mitochondria after UV irradiation (FIG. 20 b bottom twopanels). The mitochondria from UV and rottlerin-treated cells releasedmore cytochrome c than those from un-irradiated cells, but less thanthose from UV irradiated cells, indicating that the amount of PKC-δtranslocated to mitochondria decreased with rottlerin treatment.Therefore, there was a direct correlation between the amount oftranslocated PKC-δ and the released cytochrome c.

It was then studied whether PLS3 interacted with PKC-δ. PLS3 wasimmunoprecipitated from the cell lysates of UV-irradiated andnon-irradiated HEK293 cells. The precipitates were analyzed byimmunoblotting for PLS3 and for PKC-δ. PCK-δ was observed in the blot,indicating that PLS3 and PKC-δ co-immunoprecipitate. Further, thisco-immunoprecipitation increased in cells following UV irradiation (FIG.20 a, bottom panel). However, the amount of the PKC-δ thatco-immunoprecipitated with PLS3 is only about 5-10% of total cellularPKC-δ based on the estimation from a PKC-δ blot for comparison (notshown). This was not surprising since only a portion of cellular PKC-δtranslocates to mitochondria during apoptosis.

Because PKC-δ also interacts with c-abl kinase and phosphorylates c-ab1,the same IP blot was probed with c-abl antibody to determine whetherPKC-δ, PLS3, and c-abl formed a complex. However, no c-abl signal wasdetected in the immunoprecipitates of PLS3 (not shown). The fact thatPLS3 was not phosphorylated at the tyrosine residue also argues againstPLS3 as a substrate of c-abl kinase.

PKC-δ Phosphorylates PLS3 In Vitro.

Next PLS3 was evaluated as a substrate for PKC-δ using recombinantproteins. The PLS3 protein was a high-affinity substrate for PKC-δ andthe phosphorylation steadily increased over the first 25 minutes (FIG.21 a). Various concentrations of PLS3 were tested, and it was determinedthat the km is 10.5 nM (FIGS. 21 b,c). This is a very high affinity forPKC-δ toward PLS3 compared with other physiologic substrates of the PKCfamily.

Phorbol ester PMA induces apoptosis in cells overexpressing wild-typePLS3.

If PLS3 is a direct downstream effector of PKC-δ in the PKC-δ-inducedapoptotic pathway, then overexpression of PLS3 might enhance apoptosisinduced by PKC-δ activation. HeLa-PLS3 were incubated with phorbol esterPMA, and analyzed for apoptosis with TUNEL assay and flow cytometry. PMAtreatment of the control HeLa cells did not induce any apoptosis, buttreatment of the HeLa-PLS3 cells shifted the curve to right (FIG. 22).To eliminate the effect of classic PKCs (a, 3, and y) that are alsostimulated by PMA, they were treated with a combination of PMA and theindolocarbazole Go6976, which is a potent inhibitor for PKC-α, β, and γ,but does not affects PKC-δ. The curve of HeLa-PLS3 cells had a similarshift to right like that of PMA alone, indicating that this apoptoticeffect by PMA is not related with the classic PKCs. These data supportthat the overexpression of the PKC-δ substrate PLS3 makes themitochondrial effect of PKC-δ so dominant to induce apoptosis.

A mutant of PLS3 was also constructed by mutating Phe258 to Val. ThisPhe258 is in a highly conserved calcium-binding motif, and mutation ofthe corresponding Phe at PLS1 completely abolishes the activity of PLS1.Overexpressing PLS3(F258V) mutant in HeLa cells resulted in a higherbaseline apoptosis probably due to interfering with mitochondrialfunctions. PMA treatment of HeLa-PLS3(F258V) cells decreased apoptosisby shifting the curve to left (FIG. 22, bottom panel). When the samecells were treated with both PMA and Go6976, the left shift by PMA wasinhibited, indicating that the left shift was due to activation of asurvival signal from a classic PKC-related pathway.

Mitochondrial targeted PKC-δ Induced Apoptosis in HeLa-PLS3 Cells.

Because PMA activates many other isoforms of PKCs and does not representPKC-δ-specific stimulation, a mitochondrial targeted PKC-δ wasconstructed by inserting the mitochondrial targeting sequence ofcytochrome oxidase at the N-terminus of PKC-δ. The construct wastransfected into HeLa cells and G418-resistant clones were selected. Noclones were obtained after 10 days of selection; while a controlconstruct replacing PKC-δ with EGFP resulted in many clones with EGFPexpressed in the mitochondria (not shown). This indicates thatoverexpression of PKC-δ in the mitochondria is toxic to cells. Themitochondrial targeted PKC-δ was expressed into HeLa-control, HeLa-PLS3and HeLa-PLS3(F258V) cells, and studied apoptosis with annexinV-PE 36hours after transfection. Expression of the mitochondrial targeted PKC-δinduced 17.3% of cell death in the control HeLa cells, 45.3% inHeLa-PLS3 cells, indicating that PLS3 enhanced the apoptotic effect ofPKC-δ in the mitochondria. HeLa cells expressing PLS3(F258V) had only24.9% of apoptosis in a similar study, far less effective in enhancingapoptosis than HeLa-PLS3 cells (FIG. 23 a).

To prove that the PKC-δ was expressed in the mitochondria, HeLa cellswere stained 3 days after transfection with PKC-δ antibody andco-localized with MitoTracker Red dye. In the vector-transfected cells,PKC-δ was diffusely present in the cytosol and perimembranous area, anddid not overlay with MitoTracker Red (FIG. 23 b). MitoTracker Redstaining of the Mito-PKC-δ-transfected cells revealed all mitochondriaclustered around the perinuclear area, a pattern reminiscent of earlyapoptosis. PKC-δ staining of the Mito-PKC-δ-transfected cells displayedmore PKC-δ in the perinuclear area which were overlaid with theMitoTracker Red dye. This represents the overexpressed mitochondrialtargeted PKC-8. There was also a less abundant cytosolic signal, likelyfrom the endogenous PKC-δ (FIG. 23 c).

Discussion:

PLS3 is the substrate of PKC-δ in the mitochondria. Overexpression ofPLS3 sensitizes cells to apoptotic stimuli, presumably throughaugmentation of the effect of PKC-δ. This is best supported by theinduction of apoptosis in HeLa-PLS3 cells with PMA. In the normalsituation, PMA activates many different PKCs, which lead to activationof both cell survival and death signals. Depending on which pathwayprevails, cells will become live or dead. When PLS3 is overexpressed,the PKC-δ-related PLS3 activation becomes dominant to induce apoptosis.This effect was not affected by a potent classic PKC inhibitor Go6976,confirming that it is indeed related with PKC-δ. The inactivePLS3(F258V) mutant failed to enhance the apoptotic effect of PKC-δ, andPMA actually protected HeLa-PLS3(F258V) cells through losing thedownstream effector of apoptotic PKC-δ and activation of survivalsignals induced by PMA. This was confirmed by the addition of Go6976,which inhibited the survival effect of PMA.

It is impossible to state that PLS3 is the only substrate of PKC-δ inthe mitochondria. If so, blocking PLS3 with the dominant negative mutantPLS3(F258V) would completely prevent apoptosis induced by themitochondria targeted PKC-8, which appeared not the case. However, thefact that the cells expressing PLS3(F258V) had a high baseline celldeath makes the interpretation difficult, because they may die from theoverexpressed PKC-δ in the mitochondria, or they die from poormitochondrial functions. It is impossible to rule out the possibilitythat PKC-δ phosphorylates other substrate in mitochondria to induceapoptosis. Another question is whether PKC-δ is the only kinase thatphosphorylates PLS3. The answer is likely negative as well. The factthat PLS3 has a baseline phosphorylation even before the induction ofapoptosis (FIG. 20 a) indicates that PLS3 may be phosphorylated by otherkinases, but the UV-enhanced phosphorylation is likely associated withfurther increased PLS3 activity. With the important role of PLS3 inmitochondria apoptosis, understanding the mechanism of PLS3 regulationis very important. PKC-δ is the kinase that contributes to theactivation of PLS3.

It is interesting that both PLS1 and PLS3 are phosphorylated by PKC-δduring apoptosis but at different locations. Although the significanceof PLS1 phosphorylation is unclear, it has been proposed that PLS1 mightbe related with the translocation of PS to the outer leaflet of plasmamembrane. PLS3, present in the mitochondria, translocates cardiolipin tothe outer membrane of the mitochondria, an interesting analogy when youconsider that mitochondria are evolved from a prokaryote trapped insideof an eukaryote cell in the early stage of evolution. The phospholipidscramblase family is highly conserved in almost all eulcaryotes,including yeast, Drosophila and C. elegans. Genetic studies in thoselower organisms will be very helpful to dissect the mechanism of PLSregulation.

The present invention may be embodied in other specific forms withoutdeparting from its structures, methods, or other essentialcharacteristics as broadly described herein and claimed hereinafter. Thedescribed embodiments are to be considered in all respects only asillustrative, and not restrictive. The scope of the invention is,therefore, indicated by the appended claims, rather than by theforegoing description. All changes that come within the meaning andrange of equivalency of the claims are to be embraced within theirscope.

1. A method of increasing the resistance of a cell to apoptosiscomprising the step of: inhibiting expression or activity of PLS3 in thecell.
 2. The method of claim 1, wherein inhibiting expression oractivity of PLS3 in the cell includes introducing a non-functional PLS3into the cell.
 3. The method of claim 2, wherein the sequence of thenon-functional PLS-3 is developed from PLS3α (SEQ ID NO: 1) or PLS3β(SEQ ID NO: 3).
 4. The method of claim 3, wherein the non-functionalPLS3 includes a mutation in its calcium-binding motif.
 5. The method ofclaim 4, wherein the non-functional PLS3 includes the sequence of SEQ IDNO:
 12. 6. The method of claim 4, wherein the non-functional PLS3includes the sequence of SEQ ID NO:
 13. 7. The method of claim 2,wherein the non-functional PLS3 is introduced into the cell bytransfecting the cell with a vector expressing a dominant negativenon-functional PLS-3.
 8. The method of claim 7, wherein the sequence ofthe non-functional PLS3 is developed from PLS3α (SEQ ID NO: 1) or PLS3β(SEQ ID NO: 3).
 9. The method of claim 8, wherein the non-functionalPLS3 includes a mutation in its calcium-binding motif.
 10. The method ofclaim 9, wherein the non-functional PLS3 includes the sequence of SEQ IDNO:
 12. 11. The method of claim 9, wherein the non-functional PLS3includes the sequence of SEQ ID NO:
 13. 12. The method of claim 1,wherein the step of: inhibiting expression or activity of PLS3 in thecell includes exposing the cell to an inhibitory oligonucleotide.
 13. Amethod of inducing cellular apoptosis comprising the step of increasingthe amount of PLS3 present in a cell.
 14. The method of claim 13,wherein inducing cellular apoptosis includes introducing PLS3 into thecell.
 15. The method of claim 14, wherein the sequence of the PLS-3 iseither PLS3α (SEQ ID NO: 1) or PLS3β (SEQ ID NO: 3).
 16. The method ofclaim 13, wherein the non-functional PLS-3 is introduced into the cellby transfecting the cell with a vector capable of expressing PLS-3. 17.The method of claim 16, wherein the sequence of the PLS-3 expressed iseither PLS3α (SEQ ID NO: 1) or PLS3β (SEQ ID NO: 3).
 18. An isolatednucleic acid comprising a sequence that encodes a polypeptide with theamino acid sequence of SEQ ID NO:
 1. 19. The isolated and purifiednucleic acid of claim 1, comprising the sequence of SEQ ID NO: 2 or adegenerate variant of SEQ ID NO:
 2. 20. An isolated nucleic acidcomprising a sequence that encodes a polypeptide having the sequence ofSEQ ID NO: 1, or SEQ ID NO: 1 with conservative amino acidsubstitutions.
 21. A method of identifying a compound that modulates thefunction of PLS3 in a cell, the method comprising the steps of:providing a cell expressing PLS3, contacting the cell with a testcompound, and determining whether the test compound modulates theexpression of PLS3, wherein the induction of apoptosis is an indicationthat the test compound upregulates PLS3 or interferes with its function.22. A method of identifying a compound that modulates the function ofPLS3 in a cell, the method comprising the steps of: providing a cellexpressing PLS3, contacting the cell with a test compound, exposing thecell to an apoptosis-inducing stimulus, and determining whether the testcompound modulates the expression of PLS3, wherein resistance toapoptosis is an indication that the test compound downregulates PLS3 orinterferes with its function.